Synthetic peptide compounds and methods of use

ABSTRACT

The present invention provides synthetic peptide compounds and uses thereof for therapy and diagnostics of complement-mediated diseases, such as inflammatory diseases, autoimmune diseases, and microbial and bacterial infections; and non-complement-mediated diseases, such cystic fibrosis and various acute diseases. The invention is directed to modifications of a synthetic peptide of 15 amino acids from the Polar Assortant (PA) peptide, which is a scrambled peptide derived from human Astrovirus protein. In some embodiments, the invention is directed to peptide compounds that are peptide mimetics, peptide analogs and/or synthetic derivatives of PA (e.g., sarcosine derivatives) having, for example, internal peptide substitutions, and modifications, including PEGylation at the N-terminus and C-terminus. The invention further provides methods of selecting at least one synthetic peptide for treating various conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/738,786, filed Dec. 21, 2017, which is a U.S. national phase ofInternational Application No. PCT/US2016/039421, filed Jun. 24, 2016,which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/185,202, filed Jun. 26, 2015, the entirecontents of which is hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under the VirginiaInnovation Partnership i6 funding mechanism (Sub-Award #GG11598142515),awarded by the U.S. Department of Commerce. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 30, 2019, isnamed 251110000070seqlist.txt, and is 17,033 bytes in size.

FIELD

The invention relates to synthetic peptide compounds and uses thereoffor therapy and diagnostics, including for complement-mediated diseases,such as inflammatory diseases, autoimmune diseases, and microbial andbacterial infections; and non-complement-mediated diseases, such cysticfibrosis and various acute diseases.

BACKGROUND

The Complement System

The complement system, an essential component of the innate immunesystem, plays a critical role as a defense mechanism against invadingpathogens, primes adaptive immune responses, and helps remove immunecomplexes and apoptotic cells. Three different pathways comprise thecomplement system: the classical pathway, the lectin pathway andalternative pathway. C1q and mannose-binding lectin (MBL) are thestructurally related recognition molecules of the classical and lectinpathways, respectively. Whereas IgM or clustered IgG serve as theprincipal ligands for C1q, MBL recognizes polysaccharides such asmannan. Ligand binding by C1q and MBL results in the sequentialactivation of C4 and C2 to form the classical and lectin pathwayC3-convertase. In contrast, alternative pathway activation does notrequire a recognition molecule, but can amplify C3 activation initiatedby the classical or lectin pathways. Activation of any of these threepathways results in the formation of inflammatory mediators (C3a andC5a) and the membrane attack complex (MAC), which causes cellular lysis.

While the complement system plays a critical role in many protectiveimmune functions, complement activation is a significant mediator oftissue damage in a wide range of autoimmune and inflammatory diseaseprocesses. (Ricklin and Lambris, “Complement-targeted therapeutics.” NatBiotechnol 2007; 25(11): 1265-75).

A need exists for complement regulators. On the one hand, the complementsystem is a vital host defense against pathogenic organisms. On theother hand, its unchecked activation can cause devastating host celldamage. Currently, despite the known morbidity and mortality associatedwith complement dysregulation in many disease processes, includingautoimmune diseases such as systemic lupus erythematosus, myastheniagravis, and multiple sclerosis, only two anti-complement therapies haverecently been approved for use in humans: 1) purified, humanC1-Inhibitor licensed for use in patients suffering from hereditaryangioedema (HAE) and 2) eculizumab (Soliris™), a humanized, long-actingmonoclonal antibody against C5 used in the treatment of paroxysmalnocturnal hemoglobinuria (PNH). Both PNH and HAE are orphan diseases inwhich very few people are afflicted. Currently, no complement regulatorsare approved for the more common disease processes in which dysregulatedcomplement activation plays a pivotal role. Dysregulated complementactivation can play a role in both chronic disease indications and acutedisease indications. Acute disease indications include, amongst others,acute intravascular hemolytic transfusion reaction (AIHTR), birthasphyxia, hypoxic ischemic encephalopathy, ischemia-reperfusion injury(IRI) in myocardial infarct, coronary artery bypass surgery and stroke,and solid organ transplantation rejection.

Astrovirus Coat Protein

The Astroviridae constitute a family of non-enveloped, icosahedralviruses with a single-stranded, messenger-sense RNA genome. Theseviruses are a significant cause of gastroenteritis in humans as well asother diseases in other animal species. It is estimated that they causean estimated 2-17% of children's diarrheal illness worldwide.

The Astrovirus coat protein (“CP”) reduces the activity of thecomplement system, suggesting that the ‘active’ portion of the proteinmay have clinical utility in decreasing tissue damage fromcomplement-mediated diseases. The wild type coat protein (“WT CP”)purified from human astrovirus type 1 (HAstV-1) can bind C1q and MBL,and regulates both classical and lectin pathway activation (Bonaparte etal., 2008. J. Virol. 82, 817-827; Hair et al., 2010. Molec. Immunol. 47,792-798). This property is analogous to the properties described forhuman neutrophil peptide-1 (HNP-1)(Van Den Berg et al., 1998. Blood. 92,3898-3903; Groeneveld et al., 2007. Molec. Immunol. 44, 3608-3614). TheHAstV-1 coat protein is a 787 amino acid molecule that has beenexpressed from a recombinant baculovirus construct and then purified(Bonaparte et al., 2008. J. Virol. 82, 817-827).

Developing peptide compounds to inhibit classical, lectin andalternative pathways of the complement system is needed, as each ofthese three pathways have been demonstrated to contribute to numerousautoimmune and inflammatory disease processes. Specific blockade ofclassical and lectin pathways is particularly needed, as both of thesepathways have been implicated in ischemia reperfusion-induced injury inmany animal models. Humans with alternative pathway deficiencies suffersevere bacterial infections. Thus, a functional alternative pathway isessential for immune surveillance against invading pathogens.

Microbial and Bacterial Infections

Many microorganisms are resistant to currently available antibiotics.Current antibiotics are derived from other microbial organisms thatbacteria have competed against for space and energy over many years,which has led to rapid and predictable emergence of resistance. Some ofthe most pathogenic bacteria to humans are Pseudomonas aeruginosa,Staphylococcus aureus, MRSA, and carbapenemase-resistantenterobacteriacea (CREs) such as Klebsiella pneumonia. Pseudomonasaeruginosa and Staphylococcus aureus are also major pathogens in cysticfibrosis lungs. Gardnerella is a Gram variable, anaerobic coccobacillusthat is a common cause of bacterial vaginosis. Gardnerella also causescomplement activation and inflammation, which causes the symptoms ofbacterial vaginosis and increases the risk of HIV transmission bydisrupting normal barrier defenses in the vagina. There is a need fortreatments for bacterial vaginosis that kill the causative organism andblock inflammation. There is also a need for novel antimicrobialcompounds given the increasing resistance to conventional antibiotics.

Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) areviruses responsible for causing herpes. Infection with HSV-1 can happenfrom general interactions such as eating from the same utensils, sharinglip balm, or kissing. The virus is highly contagious, and it is possibleto get genital herpes from HSV-1 if the individual has had cold soresand performed sexual activities during that time. Similarly, HSV-2 isalso highly contagious. HSV-2 is contracted through forms of sexualcontact with a person who has HSV-2. It is estimated that around 20percent of sexually active adults within the United States have beeninfected with HSV-2, according to the American Academy of Dermatology(AAD). While HSV-2 infections are spread by coming into contact with aherpes sore, the AAD reports that most people get HSV-1 from an infectedperson who is asymptomatic, or does not have sores. Current treatmentslike acyclovir for HSV-1 or HSV-2 may not be fully efficacious. Thus,there is a need for anti-viral treatments for HSV-1 and HSV-2 that aremore effective against viruses.

Lactobacillus Growth

Lactobacillus is a genus of bacteria that contains over 180 species.Multiple Lactobacillus species are often administered together as asingle probiotic agent. In combination, various Lactobacillus specieshave been known to help individuals with irritable bowel syndrome,prevent necrotizing enterocolitis, and other neonatal infections.Because of its many health benefits, there is a need for compounds thatcan stimulate growth of Lactobacillus species.

Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disease caused by mutations in thecystic fibrosis transmembrane conductance regulator (CFTR) gene.Patients with CF produce unusually thick, sticky mucus that clogs thelungs and leads to life-threatening lung infections, and obstructs thepancreas and stops natural enzymes from helping the body break down foodand absorb vital nutrients. CF is characterized by a cycle of smallairway obstruction, infection with bacterial pathogens, e.g., P.aeruginosa, Staphylococcus aureus (including methicillin-resistant S.aureus (MRSA), Burkholderia cepacia, and inflammatory lung damage.Complement-mediated inflammation may be a major contributor toinflammatory lung damage in CF. Treatment of CF includes a regulartreatment routine to maintain lung health and good nutrition. Othertreatments for CF include airway clearance every day to help loosen andclear thick mucus that can build up in the lungs, inhaled medicines,including antibiotics, to help keep the airways clear, and pancreaticenzyme supplements to improve absorption of vital nutrients. There is aneed for both anti-inflammatory and anti-microbial treatments for CF.

Hemolytic Transfusion Reactions

Blood transfusions can be life-saving, but can also carry the risk of avariety of reactions, some of which are potentially life-threatening,such as acute intravascular hemolytic transfusion reactions (AIHTRs).AIHTR is estimated to occur in one-fifth of total transfusions.Individuals receiving frequent blood transfusions will developalloantibodies and autoantibodies to red blood cell (RBC) antigens overtime, making cross-matching increasingly difficult and thus increasingthe risk of AIHTR. Current transfusion safe-guards include “typing”,“antibody screening”, as well as “cross matching.” While these measureshave made transfusions safer, transfusion reactions still occur. AIHTRoccurs when host antibodies bind to the transfused erythrocytes,initiating classical complement pathway activation, which leads to thegeneration of the inflammatory mediators C3a and C5a, as well as C3bopsonization and hemolysis of the transfused cells via the membraneattack complex (MAC). To date, only one case has been reporteddescribing clinical intervention of an AIHTR by inhibiting generation ofthe complement anaphylatoxins C3a and C5a. No specific interventions forthese reactions exist; current management of the reaction is supportivein nature. While existing safeguards make ABO incompatibility rare inthe developed world, individuals with sickle cell disease and severethalassemias requiring frequent transfusions are at increased risk fortransfusion reactions due to the accumulation of antibodies againstminor antigenic determinates on erythrocytes. Neonatal ABOincompatibility in newborns can lead to jaundice, and, in severe cases,kernicterus. No blood banking organization or transfusion medicinepractice has a method to directly evaluate risk for complement-mediatedred blood cell lysis between donor and recipient. There is currently noeffective medical intervention for ATRs except for stopping thetransfusion. Diagnostic tools, prophylactic treatments to prevent AIHTR,and rescue treatments during an AIHTR are needed.

Birth Asphyxia

Birth asphyxia is a medical condition resulting from deprivation ofoxygen to a newborn that causes brain damage. Hypoxic ischemicencephalopathy (HIE) is a condition that occurs when the entire brain isdeprived of an adequate oxygen supply, but the deprivation is not total.HIE is most often associated with birth asphyxia. Reperfusion injury isthe tissue damage caused when blood supply returns to a tissue after aperiod of ischemia or lack of oxygen. The absence of oxygen andnutrients from blood during the ischemic period creates a condition inwhich the restoration of circulation results in inflammation andoxidative damage through the induction of oxidative stress rather thanrestoration of normal function. Complement activation is instrumental inthe development of ischemia-reperfusion injuries such as neonatal HIE.Therapeutic hypothermia (HT), the current standard of care for HIE,offers only an 11% reduction in death or disability. Published data hasshown that HT paradoxically increases pro-inflammatory complementactivation, which potentially limits its benefit. There is a need fortreatments for HIE and birth asphyxia that regulate complement and haveneuroprotective effects.

Autoimmune Hemolytic Anemia

Autoimmune hemolytic anemia (AIHA) is a disease that occurs whenantibodies directed against a person's own red blood cells cause them toburst, leading to insufficient plasma concentration. AIHA is mostcommonly caused by IgG and IgM. IgM is a potent activator of theclassical complement pathway. Thus, AIHA is characterized by complementmediated lysis of red blood cells. Thus, there is a need for therapiesdirected at the complement system to treat AIHA.

It would be desirable to develop peptide compounds that can regulatecomplement activation and can be used therapeutically to prevent andtreat complement-mediated diseases, such as inflammatory and autoimmunediseases. It would also be desirable to develop peptide compounds thattreat acute diseases and conditions such as acute intravascularhemolytic transfusion reaction (AIHTR), birth asphyxia, autoimmunehemolytic anemia, viral infections, and bacterial infections, amongstothers. It would also be desirable to develop peptide compounds thattreat cystic fibrosis.

SUMMARY

In one aspect, the present invention provides synthetic peptidecompounds that regulate the complement system and methods of using thesecompounds. Specifically, in some embodiments, the synthetic peptidecompounds can bind, regulate and inactivate C1 and MBL, and thereforecan efficiently inhibit classical and lectin pathway activation at itsearliest point while leaving the alternative pathway intact. Thesepeptide compounds are of therapeutic value for selectively regulatingand inhibiting C1 and MBL activation without affecting the alterativepathway and can be used for treating diseases mediated by dysregulatedactivation of the classical and lectin pathways. In other embodiments,the peptide compounds regulate classical pathway activation but notlectin pathway activation. The peptide compounds are useful for varioustherapeutic indications—even for indications that are unrelated tocomplement regulation. In some embodiments, these peptide compounds areof therapeutic value for treating acute diseases and conditions such asacute intravascular hemolytic transfusion reaction (AIHTR), birthasphyxia, as well as bacterial, viral, and antimicrobial infections,amongst others. These peptide compounds are also of therapeutic valuefor treating cystic fibrosis.

In some embodiments, the invention is based on the identification andmodification of peptides of 15 amino acids from Polar Assortant (PA)peptide (SEQ ID NO: 3), derivatives of the peptides, and methods oftheir use. The PA peptide is a scrambled peptide derived from humanastrovirus protein, called CP1 (SEQ ID NO: 1). The PA peptide is alsoknown as PIC1 (Peptide Inhibitors of Complement C1), AstroFend, AF, orSEQ ID NO: 3. As used herein, the term “PIC1 peptides” include SEQ IDNO:3 as well as other amino acid sequences that are the same as SEQ IDNO:3 but with PEGylation modifications. The PIC1 peptide was originallynamed as such because it was found to be associated with diseasesmediated by the complement system. In some aspects, surprisingly, theinvention is also related to use of the PA peptide and derivatives fortreating diseases and conditions that are not associated with thecomplement system, such as, but not limited to, cystic fibrosis andchronic obstructive pulmonary disease (COPD). In some embodiments, theinvention is based on the PIC1 peptide and modifications thereof, havingthe amino acid sequences and modifications as set forth in SEQ ID NOs:3-47. These peptides can be used to regulate complement activation,including complement inhibition; treat and/or prevent hemolyticreactions; treat hypoxic ischemic encephalopathy; treat cystic fibrosis;for anti-microbial use; and to treat and/or prevent other diseases andconditions disclosed herein.

In some aspects, the invention is directed to peptide compounds that arepeptide mimetics, peptide analogs and/or synthetic derivatives of PAhaving, for example, internal peptide deletions and substitutions,deletions and substitutions at the N terminus and C terminus, and thatare able to regulate the classical and lectin pathway activation bybinding to C1q and MBL.

A further embodiment of the invention is any one of the peptidecompounds of this invention, wherein the peptide compound is modifiedthrough sarcosine substitution, alanine substitution, and/or PEGylationof the N terminus, C terminus, or N terminus and C terminus.

In some embodiments, the peptide sequence has at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NOs: 3-47.

In one aspect, the invention provides a method of treating and/orpreventing a hemolytic reaction in a subject in need thereof comprising:administering to the subject in need thereof a composition comprising atherapeutically effective amount of a synthetic peptide comprising atleast about 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thesynthetic peptide has the amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47. In other embodiments, thehemolytic reaction is selected from the group consisting of acuteintravascular hemolytic transfusion reaction (AIHTR), transfusionrelated acute lung injury (TRALI), and platelet transfusionrefractoriness. In further embodiments, the composition is administeredbefore the subject is administered a blood transfusion, after thesubject is administered the blood transfusion, and/or during the bloodtransfusion.

In another aspect, the invention provides a method of treating hypoxicischemic encephalopathy in a subject in need thereof comprising:administering to the subject in need thereof a composition comprising atherapeutically effective amount of a synthetic peptide comprising atleast about 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thesynthetic peptide has the amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47.

In another aspect, the invention provides a method of treating cysticfibrosis in a subject in need thereof comprising: administering to thesubject in need thereof a composition comprising a therapeuticallyeffective amount of a synthetic peptide comprising at least about 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3-47. In some embodiments, the syntheticpeptide has the amino acid sequence and modifications selected from thegroup consisting of SEQ ID NO: 3-47.

In another aspect, the invention provides a method of treating abacterial infection in a subject in need thereof comprising:administering to the subject in need thereof a composition comprising atherapeutically effective amount of a synthetic peptide comprising atleast about 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thesynthetic peptide has the amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thebacterial infection is caused by bacteria selected from the groupconsisting of Staphylococcus aureus, Klebsiella pneumonia, Pseudomonasaeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, andGardnerella sp. In further embodiments, the bacterial infection iscaused by Gram-positive or Gram-negative bacteria.

In another aspect, the invention provides a method of enhancingLactobacillus growth in a subject comprising: administering to thesubject a composition comprising a therapeutically effective amount of asynthetic peptide comprising at least about 90% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO:3-47. In some embodiments, the synthetic peptide has the amino acidsequence and modifications selected from the group consisting of SEQ IDNO: 3-47.

In another aspect, the invention provides a method of treating a viralinfection in a subject in need thereof comprising: administering to thesubject in need thereof a composition comprising a therapeuticallyeffective amount of a synthetic peptide comprising at least about 90%sequence identity to an amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thesynthetic peptide has the amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, theviral infection is caused by herpes simplex virus 1 (HSV-1) or herpessimplex virus 2 (HSV-2).

In another aspect, the invention provides a method of treatingautoimmune hemolytic anemia in a subject in need thereof comprising:administering to the subject in need thereof a composition comprising atherapeutically effective amount of a synthetic peptide comprising atleast about 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3-47. In some embodiments, thesynthetic peptide has the amino acid sequence and modifications selectedfrom the group consisting of SEQ ID NO: 3-47. The autoimmune hemolyticanemia may be characterized by one or more of elevated serum bilirubin,excess urinary urobilinogen, reduced plasma haptoglobin, raised serumlactic dehydrogenase (LDH), hemosiderinuria, methemalbuminemia,spherocytosis, reticulocytosis, and/or erythroid hyperplasia of the bonemarrow.

In another aspect, the invention provides a method of treating birthasphyxia in a subject in need thereof comprising: administering to thesubject in need thereof a composition comprising a therapeuticallyeffective amount of a synthetic peptide comprising at least about 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3-47. In some embodiments, the syntheticpeptide has the amino acid sequence and modifications selected from thegroup consisting of SEQ ID NO: 3-47. In some embodiments, presence ofbirth asphyxia is characterized by an Apgar score of 3 or under thatlasts five minutes or more.

In another aspect, the invention provides a method of treating acutekidney injury in a subject in need thereof comprising: administering tothe subject in need thereof a composition comprising a therapeuticallyeffective amount of a synthetic peptide comprising at least about 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3-47. In some embodiments, the syntheticpeptide has the amino acid sequence and modifications selected from thegroup consisting of SEQ ID NO: 3-47.

In another aspect, the invention is a synthetic peptide comprising atleast about 90% sequence identity to an amino acid sequence of SEQ IDNO: 3-47. In some embodiments, a pharmaceutical composition can comprisea therapeutically effective amount of the synthetic peptide and at leastone pharmaceutically acceptable carrier, diluent, or excipient.

In another aspect, the invention is a synthetic peptide comprising theamino acid sequence and modifications of SEQ ID NO: 3-47. In someembodiments, the invention is a synthetic peptide comprising the aminoacid sequence and modifications of SEQ ID NO: 4-18 and 30-47. In someembodiments, the invention is a synthetic peptide comprising the aminoacid sequence and modifications of SEQ ID NO: 3 and 19-29. In someembodiments, the invention is a synthetic peptide comprising the aminoacid sequence and PEGylation modifications of SEQ ID NO: 21. In someembodiments, a pharmaceutical composition can comprise a therapeuticallyeffective amount of the synthetic peptide and at least onepharmaceutically acceptable carrier, diluent, or excipient.

In another aspect, the invention is directed to a PIC1 peptide, whereinthe peptide is modified through PEGylation of the N terminus, the Cterminus, or both the N terminus and C terminus. In another aspect, theinvention is directed to a PIC1 peptide, wherein the peptide has beenmodified by sarcosine and/or alanine substitutions. In otherembodiments, the peptide is modified through PEGylation and sarcosineand/or alanine substitutions. In some embodiments, the peptides areisolated and/or purified.

In one aspect, the invention provides a method of selecting at least onepeptide for treating a subject having cystic fibrosis comprising: (a)testing peptides selected from the group consisting of SEQ ID NOs: 3-47for activity on cystic fibrosis; and (b) selecting from the groupconsisting of SEQ ID NOs: 3-47 at least one synthetic peptide havingactivity on cystic fibrosis.

In another aspect, the invention provides a method of selecting at leastone peptide for treating a subject having a hemolytic reactioncomprising: (a) testing peptides selected from the group consisting ofSEQ ID NOs: 3-47 for activity on the hemolytic reaction; and (b)selecting from the group consisting of SEQ ID NOs: 3-47 at least onesynthetic peptide having activity on the hemolytic reaction. In someembodiments, the hemolytic reaction is selected from the groupconsisting of AIHTR, transfusion related acute lung injury (TRALI), andplatelet transfusion refractoriness.

In another aspect, the invention provides a method of selecting at leastone peptide for treating a subject having a bacterial infectioncomprising: (a) testing peptides selected from the group consisting ofSEQ ID NOs: 3-47 for activity on the bacterial infection; and (b)selecting from the group consisting of SEQ ID NOs: 3-47 at least onesynthetic peptide having activity on the bacterial infection. In someembodiments, the bacterial infection is caused by bacteria selected fromthe group consisting of Staphylococcus aureus, Klebsiella pneumonia,Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis,and Gardnerella sp. In some embodiments, the bacterial infection iscaused by Gram-positive or Gram-negative bacteria.

In another aspect, the invention provides a method of selecting at leastone peptide for enhancing growth of Lactobacillus comprising (a) testingpeptides selected from the group consisting of SEQ ID NOs: 3-47 foractivity enhancing growth of Lactobacillus species; and (b) selectingfrom the group consisting of SEQ ID NOs: 3-47 at least one syntheticpeptide having activity enhancing growth of Lactobacillus species.

In another aspect, the invention provides a method of selecting at leastone peptide for treating and/or preventing a viral infection comprising(a) testing peptides selected from the group consisting of SEQ ID NOs:3-47 for activity that treats or prevents the viral infection; and (b)selecting from the group consisting of SEQ ID NOs: 3-47 at least onesynthetic peptide having activity that treats or prevents the viralinfection. In some embodiments, the viral infection is caused by HSV-1or HSV-2.

In another aspect, the invention provides a method of selecting at leastone peptide for treating and/or preventing autoimmune hemolytic anemiacomprising (a) testing peptides selected from the group consisting ofSEQ ID NOs: 3-47 for activity on autoimmune hemolytic anemia; and (b)selecting from the group consisting of SEQ ID NOs: 3-47 at least onesynthetic peptide having activity on autoimmune hemolytic anemia.

In another aspect, the invention provides a method or selecting at leastone peptide for treating birth asphyxia comprising (a) testing peptidesselected from the group consisting of SEQ ID NOs: 3-47 for activity onbirth asphyxia; and (b) selecting from the group consisting of SEQ IDNOs: 3-47 at least one synthetic peptide having activity on birthasphyxia.

In another aspect, the invention provides a method of selecting at leastone peptide for treating hypoxic ischemic encephalopathy comprising: (a)testing peptides selected from the group consisting of SEQ ID NOs: 3-47for activity on hypoxic ischemic encephalopathy; and (b) selecting fromthe group consisting of SEQ ID NOs: 3-47 at least one synthetic peptidehaving activity on hypoxic ischemic encephalopathy.

In other aspects, the invention provides a method of treating a diseaseby administering the compositions described herein, wherein the diseasethat is at least partially complement mediated includes but is notlimited to: hemolytic transfusion reactions, cold-agglutinin disease,immune-complex diseases, thalassemia, sickle cell disease, ABOincompatibility, acute/hyperacute solid organ transplantation rejection,instant blood-mediated inflammatory reaction (IBMIR), solid organtransplantation warm/cold ischemia, systemic lupus erythematosus (SLE),rheumatoid arthritis, ischemia-reperfusion injury, myocardial infarct,stroke, hypoxic ischemic encephalopathy, traumatic brain injury,coronary artery bypass surgery, wound healing, cancer, Alzheimer'sdisease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH),atypical hemolytic uremic syndrome (aHUS), asthma, Crohn's disease,Sepsis syndrome/ARDS/SIRS, glomerulonephritis, lupus nephritis,anti-glomerular basement membrane disease, anti-neutrophil cytoplasmicautoantibody-induced, membranoproliferative glomerulonephritis, densedeposit disease, membranous nephropathy, IgA nephropathy, or C3glomerulopathy.

In another aspect, the invention provides a method of treating a diseaseby administering the compositions described herein, wherein the diseaseis not complement-mediated, which includes but is not limited to cysticfibrosis and chronic obstructive pulmonary disease (COPD).

Another embodiment of the invention is a method of treating a diseaseassociated with complement-mediated tissue damage, further comprisingadministering to a subject at least one other active ingredienteffective in treating the disease, wherein at least one other activeingredient includes a non-steroidal anti-inflammatory agent, acorticosteroid, a disease modifying anti-rheumatic drug, C1-inhibitor,and eculizumab.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the titration of PA-dPEG24 (SEQ ID NO: 21), PA-P7Sar (SEQID NO: 10), and PA-C9Sar (SEQ ID NO: 12), in a hemolytic assay usingfactor B depleted serum. Concentrations of peptides are shown in mM.

FIGS. 2A-B show THAT PA-dPEG24 (SEQ ID NO: 21) inhibits complementactivation in a hemolytic assay using factor B depleted serum to thesame degree as PA (SEQ ID NO: 3).

FIG. 3 shows the structure of PA-dPEG24 (SEQ ID NO: 21).

FIGS. 4A-E show plate dilution assays. FIG. 4A shows plate dilutionassays for Staphylococcus aureus treated with PA-dPEG24 (SEQ ID NO: 21)or saline control. The X-axis shows inhibitor concentration of PA-dPEG24(SEQ ID NO: 21) in mg/ml. FIG. 4B shows plate dilution assays forStaphylococcus aureus treated with PA-dPEG24 or Vancomycin. The X-axisshows inhibitor concentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml, andvancomycin starting at 5 μg/ml. FIG. 4C shows plate dilution assays forKlebsiella pneumonia treated with PA-dPEG24 (SEQ ID NO: 21) orGentamycin. The X-axis shows inhibitor concentration of PA-dPEG24 (SEQID NO: 21) in mg/ml. Gentamycin starting at 4 μg/ml. FIG. 4D shows platedilution assays for Pseudomonas aeruginosa treated with PA-dPEG24 (SEQID NO: 21) or Gentamycin control. The X-axis shows inhibitorconcentration of PA-dPEG24 (SEQ ID NO: 21) in mg/ml. Gentamycin startingat 4 μg/ml. FIG. 4E shows plate dilution assays for Pseudomonasaeruginosa treated with four different versions of PA-dPEG24 (SEQ ID NO:21) or Gentamycin control. Figure Legend: PIC1=PA-dPEG24 (SEQ ID NO:21); PA-L3Sar (H2N-IA(Sar)ILEPICCQERAA-OH) (SEQ ID NO: 6); PA-I4Sar(H2N-IAL(Sar)LEPICCQERAA-OH) (SEQ ID NO: 7); PA-L5Sar(H2N-IALI(Sar)EPICCQERAA-OH) (SEQ ID NO: 8).

FIG. 5 shows inhibition of Gardnerella growth in the presence ofPA-dPEG24 (SEQ ID NO: 21), PA-L3Sar (SEQ ID NO: 6), PA-I4Sar (SEQ ID NO:7), or PA-L5Sar (SEQ ID NO: 8). PIC1=PA-dPEG24 (SEQ ID NO: 21);Sarc-1=PA-L3Sar (SEQ ID NO: 6); Sarc-2=PA-I4Sar (SEQ ID NO: 7);Sarc-3=PA-L5Sar (SEQ ID NO: 8).

FIG. 6 shows C5a release when serum is incubated with Gardnerella atdifferent concentrations of PA-dPEG24 (SEQ ID NO: 21). PIC1=PA-dPEG24(SEQ ID NO: 21).

FIG. 7 shows two type-O plasmas from the RBC transfusions that causedATR in a blood type-B recipient. The IgG titers for each plasma wasquite high, but non-discriminatory. However, in a CH50-type hemolyticassay, they behave drastically differently. One is highly hemolytic,while the other does not cause significant hemolysis.

FIG. 8 shows the type-O plasma (#426) from the RBC transfusions thatcaused ATR in blood type-B recipient. Prior to adding B erythrocytes,PA-dPEG24 (SEQ ID NO: 21) was added to the plasma in increasingconcentrations. A dose-response inhibition of hemolysis was demonstratedwith PA-dPEG24 (SEQ ID NO: 21) demonstrating up to >95% inhibition.

FIG. 9 shows bacteria initiating classical pathway complement activationwith C5a-mediated neutrophil recruitment and activation.

FIGS. 10A-D shows complement anaphylatoxins in CF and control lungfluid. FIG. 10A: C5a concentrations in soluble (sol) fractions fromsputum of CF patients (n=15) and controls (n=3). Box shows quartiles,whiskers are 90^(th) and 10^(th) percentile, and dashed line is themean. CF sols C5a level is 5-fold higher than control sols (P=0.04).FIG. 10B: C5a Western blot for sputum sols for two healthy controls (Aand B) and 2 CF subjects (X and Y). FIG. 10C: C3a concentrations in solfractions from sputum of CF patients (n=14) and controls (n=4). Boxshows quartiles, whiskers are 90^(th) and 10¹⁰ percentile, and dashedline is the mean. P=0.03. FIG. 10D: C4a concentrations in sol fractionsfrom sputum of CF patients (n=15) and controls (n=5). Box showsquartiles, whiskers are 90^(th) and 10^(th) percentile, and dashed lineis the mean.P=0.05.

FIGS. 11A-B shows complement opsonization of Staphylococcus aureus. FIG.11A: Staphylococcus aureus-bound C3-fragments after incubation in solfractions from sputum of CF patients (n=5) and controls (n=3). Box showsquartiles, whiskers are 90^(th) percentile, and dashed line is the mean.P=0.42. FIG. 11B: Staphylococcus aureus-bound C4-fragments afterincubation in sol fractions from sputum of CF patients (n=5) andcontrols (n=3). Box shows quartiles, whiskers are 90^(th) and 10^(th)percentile, and dashed line is the mean. P=0.13.

FIGS. 12A-D shows C5a generated by bacteria in CF sols. FIG. 12A: C5aconcentrations in sol fractions from sputum of CF patients (n=3) orcontrols (n=3) before and after incubation with live or dead Pseudomonasaeruginosa or Staphylococcus aureus. Data are means±SE. C5a wasgenerated in CF sol in the presence of Pseudomonas aeruginosa (P=0.03)or Staphylococcus aureus (P=0.03). FIG. 12B: C5a concentrations in solfractions from sputum of CF patients (subjects A, B, and C) before(Initial) and after incubation with live or dead Pseudomonas aeruginosa.FIG. 12C: C5a concentrations in sol fractions from sputum of CF patients(subjects A, B, and C) before (Initial) and after incubation with liveor dead Staphylococcus aureus. FIG. 12D: C5a concentrations in CF solsthat were incubated alone in buffer (CF sol only), incubated with deadPseudomonas aeruginosa (CF sol+Pseudomonas aeruginosa), or incubatedwith PA-dPEG24 (SEQ ID NO: 21) and dead Pseudomonas aeruginosa (CFsol+PA-dPEG24+P. aerug). PA-dPEG24, SEQ ID NO: 21 reduces C5a generationin CF sols incubated with Pseudomonas aeruginosa.

FIGS. 13A-C shows correlation plots for complement effectors andclinical measures. FIG. 13A: C5a concentrations in CF sols positivelycorrelate with increasing age, r=0.53, P=0.04. FIG. 13B: C5aconcentrations in CF sols correlate inversely with BMI percentile inchildren, r=−0.77, P=0.04. FIG. 13C: C3a concentrations in CF solspositively correlate with FEV1%, rs=0.63, P=0.02.

FIGS. 14A-E shows peptide compounds binding and inhibiting C1q. FIG.14A: shows PA (SEQ ID NO: 3) binding to C1q, but not to CRT or BSA. FIG.14B shows PIC1 (SEQ ID NO: 21) inhibits DyLight 680 labeled C1q bindingin recombinant CRT. FIG. 14C shows PIC1 (SEQ ID NO: 21) inhibits DyLightlabeled 488 C1q binding to Raji cells, and FIG. 14D shows representativeIn-Cell Western plate assay imaged on LICOR Odyssey demonstrating thatincreasing amounts of PIC1 (SEQ ID NO: 21) inhibit DyLight 680 labeledC1q from binding Raji cells. FIG. 14E shows the experiment setup.

FIG. 15 shows soluble C1q binding calreticulin (CRT), but not BSA.

FIG. 16 shows PA (SEQ ID NO: 3) inhibiting C1q binding to calreticulin(CRT) compared to a negative control peptide CP2.

FIGS. 17A-C shows Raji cells at 20× magnification. FIG. 17B: FITC (C1q)at 1/50 s exposure. FIG. 17C: DAPI at 1/500 s exposure. FIG. 17A:Overlay.

FIGS. 18A-C shows Raji cells at 20× magnification treated with 0.523 mMPA-dPEG24 (SEQ ID NO: 21). FIG. 18B: FITC (C1q) at 1/50 s exposure. FIG.18C: DAPI at 1/500 s exposure. FIG. 18A: Overlay.

FIGS. 19A-C shows Raji cells at 20× magnification treated with 1.05 mMPA-dPEG24 (SEQ ID NO: 21). FIG. 19B: FITC (C1q) at 1/50 s exposure. FIG.19C: DAPI at 1/500 s exposure. FIG. 19A: Overlay.

FIG. 20 shows red blood cell lysis in a hemolytic assay in rats treatedwith two different doses of PA-dPEG24 (SEQ ID NO: 21) or with normalsaline.

FIGS. 21A-B shows the experimental design and study arms. FIG. 21A showsPA-dPEG24 (SEQ ID NO: 21) dosing in AIHTR model, FIG. 21B showsPA-dPEG24 (SEQ ID NO: 21) efficacy in AIHTR model.

FIGS. 22A-B shows PA-dPEG24 (SEQ ID NO: 21) dosing studies in AIHTRmodel comparing high dose PA-dPEG24 (SEQ ID NO: 21) (40 mg) vs. losedose PA-dPEG24 (SEQ ID NO: 21) (20 mg). FIG. 22A shows free hemoglobinpresent in rat plasma collected at 0 sec (prebleed), 30 seconds, 5minutes, 20 minutes, 60 minutes, and 120 minutes after 15% transfusionof human red blood cells (HuRBCs), measured by spectrophotometry.Vehicle control is saline. Positive control is cobra venom factor (CVF).n=number of animals in each group. Error bars denote standard error ofthe means. FIG. 22B shows percent of surviving HuRBCs (detected usingFITC-conjugated anti-human CD235a (glycophorin A monoclonal antibody) at30 seconds, 5 minutes and 20 minutes after transfusion measured by flowcytometry. Error bars denote standard error of the means. CVF: cobravenom factor. Saline: 0.9% normal saline.

FIGS. 23A-C shows PA-dPEG24 (SEQ ID NO: 21) efficacy in prophylaxis(pre-transfusion) vs. treatment (rescue therapy, given aftertransfusion) in AIHTR model. FIG. 23A shows free hemoglobin present inrat plasma collected at 0 second (pre-bleed), 30 seconds, 5 minutes, 20minutes, 60 minutes, and 120 minutes after 15% transfusion of HuRBCs,measured by spectrophotometry. Error bars denote standard error of themeans. FIG. 23B shows area under the curve (total hemoglobin released)over 120 minutes. Error bars denote standard error of the means. FIG.23C shows unconjugated bilirubin measured at 120 minutes compared topre-transfusion (pre-bleed). Error bars denote standard error of themeans.

FIGS. 24A-D shows PA-dPEG24 (SEQ ID NO: 21) protection of human RBCsfrom lysis by rat serum in vitro. FIG. 24A shows flow cytometry analysisof HuRBCs incubated for 5 min in rat serum then labeled withanti-glycophorin A (APC) and anti-C3 (FITC). Labeled opsonized RBCsspiked into unlabeled RBCs (representative plot). FIG. 24B shows flowcytometry analysis of HuRBC's incubated 5 min in rat serum treated withPA-dPEG24 (representative plot). FIG. 24C shows relative increase innumber of HuRBCs with no C3 deposition (Q3) in PA-dPEG24 treated serumcompared to untreated serum after 5 min. Error bars denote standarderror of the means for two independent experiments. FIG. 24D showspercent of serum-incubated RBCs that bound C3-fragments (Q2/Q2+Q3) isreduced in PA-dPEG24 treated serum compared to untreated serum after 5min. Error bars denote standard error of the means for 2 independentexperiments. Q2: Cells dual stained with C3 and glycophorin A (C3deposition on glycophorin A labeled cells). Q3: Cells with glycophorin Alabel (intact cells).

FIGS. 25A-F shows PA-dPEG24 (SEQ ID NO: 21) protection versus saline ofhuman RBCs transfused into rats at 30 seconds (FIGS. 25A and 25D), 5minutes (FIGS. 25B and 25E) and 20 minutes (FIGS. 25C and 25F). Flowcytometry analysis of RBCs recovered from blood draws and labeled withanti-glycophorin A (APC) and anti-C3 (FITC). In FIGS. 25A-C, the animalwas given saline control. In FIGS. 25D-F, the animal wasprophylactically treated with PA-dPEG24 (SEQ ID NO: 21). Q2: Cells dualstained with C3 and glycophorin A. Q3: Cells with glycophorin A label(intact HuRBCs).

FIGS. 26A-D show transfused human RBC survival in rats. FIG. 26A showsthe number of events measured at 30 seconds after transfusion withHuRBCs showing all anti gpA+RBCs (Q2+Q3), anti gpA+anti C3-(Q3) and antigpA+anti C3+(Q2) for PA-dPEG24 (SEQ ID NO: 21) prophylaxis, salinecontrol, CVF (cobra venom factor) control and PA-dPEG24 (rescue)treatment. Error bars denote standard error of the means. FIG. 26B showspercent of anti gpA+, anti C3+RBCs compared with total anti gpA+RBCs(Q2/Q2+Q3). Error bars denote standard error of the means. FIG. 26Cshows total anti gpA+RBCs (Q2+Q3) present in blood at 5 minutes and 20minutes compared to the controls (Saline and CVF). Error bars denotestandard error of the means. FIG. 26D shows anti gpA− anti C3+RBCs (Q1)present in blood up to 20 minutes. Legend: PIC1 (SEQ ID NO: 21)prophylaxis is denoted with a star; PIC1 (SEQ ID NO: 21) Treatment(rescue treatment compared with saline control) is denoted with adiamond; saline control is denoted with a square; CVF control is denotedwith a triangle. Error bars denote standard error of the means.

FIGS. 27A-F shows PA-dPEG24 (SEQ ID NO: 21) protection of human RBCstransfused into rats as compared to immune globulin intravenous (IVIG)at 30 seconds (FIGS. 27A and 27D), 5 minutes (FIGS. 27B and 27E) and 20minutes (FIGS. 27C and 27F) post-transfusion. Flow cytometry analysis ofRBCs recovered from blood draws and labeled with anti-glycophorin A(APC) and anti-C3 (FITC). FIGS. 27A-C show the animal prophylacticallytreated with IVIG. FIGS. 27D-F show the animal prophylactically treatedwith PA-dPEG24. Q2 shows cells dual stained with C3 and glycophorin A.Q3 shows cells with glycophorin A label (intact HuRBCs).

FIGS. 28A-C shows prophylactic PA-dPEG24 (SEQ ID NO: 21) efficacy ascompared to prophylactic immune globulin intravenous (IVIG). FIG. 28Ashows free hemoglobin present in rat plasma collected at 0 second(pre-bleed), 30 seconds, 5 minutes, 20 minutes, 60 minutes, 120 minutes,180 minutes, 240 minutes, 300 minutes, and 360 minutes after 15%transfusion of HuRBCs, measured by spectrophotometry. Error bars denotestandard error of the means. FIG. 28B shows number of events measured at30 seconds after transfusion with HuRBCs showing all anti gpA+RBCs(Q2+Q3), anti gpA+ anti C3-(Q3) and anti gpA+ anti C3+(Q2) for PA-dPEG24prophylaxis and IVIG prophylaxis. Error bars denote standard error ofthe means. FIG. 28C shows anti gpA− anti C3+ RBCs (Q1) present in bloodat 30 seconds for PA-dPEG24 prophylaxis and IVIG prophylaxis. Error barsdenote standard error of the means. FIG. 28D shows Total anti-gpA1 RBCsfor IVIG (no lines) and PIC1 (diagonal lines) prophylaxis at 5 and 20minutes after transfusion.

FIGS. 29A-C shows prophylactic PA-dPEG24 (SEQ ID NO: 21)(labeled asPIC1) efficacy as compared to prophylactic immune globulin intravenous(IVIG) on acute kidney injury. FIG. 29A shows gross kidney weightsmeasured for IVIG prophylaxis and PA-dPEG24 prophylaxis prior toformalin fixing. Error bars denote standard error of the means, n=6.FIG. 29B shows gross kidney image for IVIG prophylaxis and PA-dPEG24prophylaxis prior to formalin fixing (representative animal). FIG. 29CRepresentative histology (hematoxylin and eosin stain) of kidneys fromrats receiving saline (i, ii), IVIG prophylaxis (iii, iv), or PIC1prophylaxis (v, vi). PIC1-treated rats demonstrate normal kidneyarchitecture, whereas saline- and IVIG-treated rats show disruption ofcellular architecture consistent with acute tubular necrosis. Barrepresents 20 mm. Tissues were observed with a microscope (Bmax,Olympus) at a magnification of 4003 at room temperature. Images wereacquired with a digital camera (DP71, Olympus).

FIG. 30 shows percent maximal serum hemolysis after hypoxia in rats at1, 2, 4, 8, 16, 24, 48 hours post-treatment with PIC1 (PA-dPEG24) (SEQID NO: 21), cobra venom factor (CVF), or therapeutic hypothermia (31-32°C. for 6 hours).

FIG. 31 shows images of brain slices after hypoxia in rats treated withPIC1 peptide (PA-dPEG24), cobra venom factor (CVF) therapeutichypothermia (31-32° C. for 6 hours).

FIG. 32A shows combined C1q cranial levels in HIE. There issignificantly less C1q deposition in the PIC1 (SEQ ID NO: 21) treatedgroup (Normothermia+ PIC1 (SEQ ID NO: 21)-triangle) when compared tountreated HIE controls (Normothermia-diamond) at 4, 12, and 24 hoursafter brain injury. PIC1 (SEQ ID NO: 21) treated animals had similarlevels of C1q compared to animals who underwent therapeutic hypothermia(square).

FIG. 32B shows evidence of neuroprotection post-HIE with PIC1 (SEQ IDNO: 21) treatment. Panels A-D of FIG. 32B show cresyl violet staining.Cresyl violet stains Nissl substance in neurons. The HIE group (Panel B)shows a significant decrease in cresyl violet staining compared to thePIC1 (SEQ ID NO: 21) group (Panel D), indicating the neuro-preservatoryaction of PIC1 (SEQ ID NO: 21) similar to therapeutic hypothermia (PanelC). Panels E-H show hematoxylin and eosin staining. Hematoxylin andEosin staining demonstrated a greater degree of neuronal destruction(necrosis, pyknosis and karyorrhexis) in the HIE brain (Panel F) whencompared to those treated with PIC1 (SEQ ID NO: 21) (Panel H). PanelsI-L show acridine orange staining. Acridine orange stains viable partsof the brain a bright green (Panels K and L). HIE significantlydecreases acridine orange staining in the brain (Panel J). PIC1 (SEQ IDNO: 21) treatment restored acridine orange staining, indicating thepresence of more viable cells in the brain (Panel L).

FIG. 32C shows histological neuroprotection also translated intoneurofunctional improvement after HIE. Animals injected with PIC1 (SEQID NO: 21) performed better than normothermic animals and similar tono-intervention animals or animals receiving therapeutic hypothermia (Nointervention, first bar on the left; HIE: Normothermia, second bar fromleft; HIE+therapeutic hypothermia: Hypothermia, third bar from left,HIE+PIC1(SEQ ID NO: 21): Normo-PIC1 (SEQ ID NO: 21), fourth bar fromleft).

FIG. 33 shows PIC1 (SEQ ID NO: 21) vs. Acyclovir inhibition of HSV-1encoding GFP. In the no virus, negative control (as labeled), thehistogram shows a peak to the left, indicating that there are noinfected cells (GFP−). In the positive control, the peak shifts to theright, indicating infected cells (GFP+). In row 2, an increasingconcentration of PIC1 (SEQ ID NO: 21) gradually shifts the peak to theleft, indicating that PIC1 (SEQ ID NO: 21) inhibits viral replication.In contrast, row 3 shows the dose-dependent effect of Acyclovir (ACV) onviral replication. Treatment with 5 mM of ACV resulted in a small rightpeak, indicating that there was still residual viral replication.

FIG. 34 shows PIC1 (SEQ ID NO: 21) vs. Acyclovir inhibition of HSV-2. Inthe negative control (as labeled), the histogram shows a peak to theleft, indicating that there are no infected cells (GFP−). In thepositive control, the peak shifts to the right, indicating infectedcells (GFP+). In row 2, an increasing concentration of PIC1 (SEQ ID NO:21) gradually shifts the peak to the left, indicating that PIC1 (SEQ IDNO: 21) inhibits viral replication. In contrast, row 3 shows thedose-dependent effect of Acyclovir (ACV) on viral replication.

FIG. 35 shows that PIC1 (SEQ ID NO: 21), termed AF1 on the x-axis of thefigure, promotes the growth of L. acidophilus and L. leichmannii.

FIG. 36A-C shows the antimicrobial activity of PIC1 (SEQ ID NOS: 7, 8,12, and 21). FIG. 36A shows that PIC1 (SEQ ID NO: 21) inhibits growth ofStaphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniaein microdilution MIC testing. FIG. 36B shows peptide variants AF1-AF5(SEQ ID NO: 21, 5, 6, 7 and 8, respectively) in microdilution MICtesting for Pseudomonas aeruginosa. FIG. 36C shows confocal microscopyshowing PIC1 (SEQ ID NO: 21) bound to the outer surface of bacteria.

FIG. 37A-D shows Neisseria gonorrhoeae growth in presence or absence ofPIC1 (SEQ ID NO: 21). FIG. 37A shows Neisseria gonorrhoeae growth inpresence or absence of 20 mg/ml of PIC1 (SEQ ID NO: 21). FIG. 37B showsNeisseria gonorrhoeae growth in presence or absence of 30 mg/ml of PIC1(SEQ ID NO: 21). FIG. 37C shows Neisseria gonorrhoeae grown in titrationconcentrations of PIC1 (SEQ ID NO: 21). FIG. 37D shows colony countsfrom Neisseria gonorrhoeae incubated with and without PIC1 (SEQ ID NO:21).

FIG. 38 shows PIC1(SEQ ID NO: 21) inhibits MPO activity in sputumsamples (sol) isolated from cystic fibrosis (CF) patients. CF solsamples were incubated in the presence or absence of 20 mg/ml PIC1 (SEQID NO: 21) for 30 minutes followed by addition of TMB for 30 minutes atroom temperature. MPO activity was then measured by detection of TMBcolor change in a spectrophotometer at 450 nm.

FIG. 39 shows PIC1 (SEQ ID NO: 21) dose-dependently inhibits MPOactivity in a sputum samples (sol) isolated from a cystic fibrosis (CF)patient. The CF sol was incubated in the presence of increasing amountsof PIC1 (SEQ ID NO: 21) at room temperature. MPO activity was thenmeasured by detection of TMB color change in a spectrophotometer at 450nm.

FIG. 40 shows PIC1 (SEQ ID NO: 21) inhibits MPO activity in rat brainssubject to hypoxic ischemic encephalopathy (HIE). Supernatants from ratbrain lysate that underwent HIE was incubated in the presence or absenceof 20 mg/ml PIC1 (SEQ ID NO: 21). MPO activity was then measured bydetection of TMB color change in a spectrophotometer at 450 nm.

FIG. 41 shows brain lysates from rat pups with HIE either receiving notreatment (NT=normothermia), Hypothermia (HT), or PIC1 (SEQ ID NO: 21)i.p. (NP=normothermia+ PIC1 (SEQ ID NO: 21)). The lysates were thentested for MPO activity with TNB. Over 48 hours the NT animalsexperience increasing MPO activity consistent with increased reperfusioninjury and infarction size. The PIC1 (SEQ ID NO: 21) treated animalshows a trend towards decreased MPO activity compared with no treatment(NT) at each time point, suggesting a decrease in MPO activity in theHIE brain after i.p. administration of PIC1 (SEQ ID NO: 21).

FIG. 42 shows that PIC1 (SEQ ID NO: 21) dose-dependently inhibits MPOactivity in lysates of purified human neutrophils (PMN). PMN lysateswere incubated in the presence of increasing amounts of PIC1 (SEQ ID NO:21). MPO activity was then measured by detection of TMB color change ina spectrophotometer at 450 nm.

FIG. 43 shows titration of the inhibition of MPO activity by increasingamounts of PIC1 (SEQ ID NO: 21). PIC1 (SEQ ID NO: 21) directly inhibitsMPO.

FIG. 44 shows that increasing amounts of RBC lysates containinghemoglobin demonstrated a dose-dependent increase in the oxidation ofthe chromogen tetramethylbenzidine (TMB) substrate. In the presence ofPIC1 (SEQ ID NO: 21) at 20 mg/ml, the oxidation of TMB was inhibited.

FIG. 45 shows increasing amounts of PIC1 (SEQ ID NO: 21) led to adose-dependent decrease in oxidized TMB signal by hemoglobin from RBClysates.

FIG. 46 shows that increasing amounts of PIC1 (SEQ ID NO: 21)dose-dependently inhibited oxidation of tetramethylbenzidine TMB withincreasing amounts of hemoglobin from RBC lysate.

DETAILED DESCRIPTION

The present invention provides synthetic peptide compounds based on themodification of isolated, purified peptides of 15 amino acids from PolarAssortant (PA) peptide (SEQ ID NO: 3), derivatives of the peptides, andmethods of their use. The PA peptide is a scrambled, shortened peptidederived from human astrovirus protein, called CP1 (SEQ ID NO: 1). Insome embodiments, the invention is based on modification of an isolated,purified peptide of CP1, and the peptide derivatives have the amino acidsequences set forth in SEQ ID NOs:4-47. SEQ ID NOs: 4-47 are derivativesof PA (SEQ ID NO: 3) having, for example, sarcosine substitutions and/ormodifications at the N terminus and C terminus, including PEGylation.These peptides can be used to regulate complement activation, includingcomplement inhibition; treat and/or prevent hemolytic reactions; treathypoxic ischemic encephalopathy; treat cystic fibrosis; foranti-microbial use; and treat and/or prevent other diseases andconditions disclosed herein.

In some aspects, these peptide compounds are of therapeutic value forthe treatment of diseases and conditions mediated by dysregulatedactivation of the classical and lectin pathways. In some aspects, theinvention provides methods for selecting peptides that haveanti-microbial activity, can be used to treat cystic fibrosis, and/orcan be used to treat various acute diseases. The PA peptide (SEQ ID NO:3) has low solubility in aqueous solutions. The peptides of SEQ ID NO:21 and other PEGylated and sarcosine substitutions (e.g., SEQ ID NOs:5-8, 10-12, 21-25, 27-29, 30-40, 43, 45, and 47), have increasedsolubility in aqueous solutions, which may increase their efficacy astherapeutic compounds.

In some embodiments, the invention is based on the identification andmodification of an isolated, purified peptide of 30 amino acids derivedfrom human astrovirus coat protein, termed CP1, and having a sequence(SEQ ID NO:1) that is able to regulate the classical and lectin pathwayactivation by binding to C1q and MBL. In other embodiments, the peptidecompounds regulate the classical pathway activation but not the lectinpathway activation.

Modifications of the amino acid structure of CP1 has led to thediscovery of additional peptide compounds that are able to regulatecomplement activation, such as C1q activity.

The term “peptide compound(s),” as used herein, refers to amino acidsequences, which may be naturally occurring, or peptide mimetics,peptide analogs and/or synthetic derivatives of about 15 amino acidsbased on SEQ ID NO: 3. In addition, the peptide compound may be lessthan about 15 amino acid residues, such as between about 10 and about 15amino acid residues and such as peptide compounds between about 5 toabout 10 amino acid residues. Peptide residues of, for example, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, and 15 amino acids are equally likely to bepeptide compounds within the context of the present invention. Peptidecompounds can also be more than 15 amino acids, such as, for example,16, 17, 18, 19, and 20, or more amino acids.

The disclosed peptide compounds are generally constrained (that is, havesome element of structure as, for example, the presence of amino acidsthat initiate a β turn or β pleated sheet, or, for example, are cyclizedby the presence of disulfide bonded Cys residues) or unconstrained (thatis, linear) amino acid sequences of about 15 amino acid residues, orless than about 15 amino acid residues.

Substitutes for an amino acid within the peptide sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. Amino acids containing aromatic ringstructures include phenylalanine, tryptophan, and tyrosine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The positively charged (basic)amino acids include arginine and lysine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration.

A conservative change generally leads to less change in the structureand function of the resulting protein. A non-conservative change is morelikely to alter the structure, activity, or function of the resultingprotein. For example, the peptide of the present disclosure comprisesone or more of the following conservative amino acid substitutions:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine; replacement of a threonine with a serine;replacement of an acidic residue, such as aspartic acid and glutamicacid, with another acidic residue; replacement of a residue bearing anamide group, such as asparagine and glutamine, with another residuebearing an amide group; exchange of a basic residue, such as lysine andarginine, with another basic residue; and replacement of an aromaticresidue, such as phenylalanine and tyrosine, with another aromaticresidue.

Particularly preferred amino acid substitutions include:

-   -   a) Ala for Glu or vice versa, such that a negative charge may be        reduced;    -   b) Lys for Arg or vice versa, such that a positive charge may be        maintained;    -   c) Ala for Arg or vice versa, such that a positive charge may be        reduced;    -   d) Glu for Asp or vice versa, such that a negative charge may be        maintained;    -   e) Ser for Thr or vice versa, such that a free —OH can be        maintained;    -   f) Gln for Asn or vice versa, such that a free NH2 can be        maintained;    -   g) Ile for Leu or for Val or vice versa, as roughly equivalent        hydrophobic amino acids;    -   h) Phe for Tyr or vice versa, as roughly equivalent aromatic        amino acids; and    -   i) Ala for Cys or vice versa, such that disulphide bonding is        affected.

Substitutes for an amino acid within the peptide sequence may beselected from any amino acids, including, but not limited to alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine,tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or otherN-methylated amino acids. In some embodiments, sarcosine substitutes foran amino acid within the peptide sequence.

In one embodiment, the invention discloses synthetic peptides derivedfrom human astrovirus coat protein, the peptides comprising the aminoacid sequences and modifications of SEQ ID NOs: 3-47. In someembodiments, the invention discloses synthetic peptides derived fromhuman astrovirus coat protein, the peptides comprising the amino acidsequences and modifications of SEQ ID NOs: 3 and 19-29. In othersembodiments, the invention discloses synthetic peptides derived fromhuman astrovirus coat protein, the peptides comprising the amino acidsequences and modifications of SEQ ID NOs: 4-18 and 30-47.

In another embodiment, the invention discloses a synthetic peptidecomprising the amino acid sequence of SEQ ID NO: 3, with one or moreamino acid substitutions, modifications, insertions, or deletions,wherein the peptide regulates complement activation and/or hasanti-microbial activity, amongst other therapeutic activities describedherein.

In another embodiment, the invention discloses a synthetic peptidecomprising the amino acid sequence of SEQ ID NO: 3, with one or moresarcosine substitutions, wherein the peptide regulates complementactivation and/or has anti-microbial activity amongst other therapeuticactivities described herein.

In another embodiment, the invention discloses a synthetic peptidecomprising the amino acid sequence of SEQ ID NO: 3, with one or morealanine substitutions, wherein the peptide regulates complementactivation and/or has anti-microbial activity amongst other therapeuticactivities described herein.

In another embodiment, the invention discloses a synthetic peptidecomprising the PEGylated amino acid sequence of SEQ ID NO: 3, whereinthe peptide regulates complement activation and/or has anti-microbialactivity amongst other therapeutic activities described herein.

The peptide compounds may have internal peptide deletions andsubstitutions as well as deletions and substitutions at the N-terminusand C-terminus based on SEQ ID NO: 3. In some embodiments, the peptidehas about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions, modifications, insertions, or deletions.

In some embodiments, the peptide sequence has at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO: 3.

In some embodiments, the present invention relates to therapeuticallyactive peptides having the effects of treating or preventing organdysfunction induced by ischemia, inflammation and/or toxic effects ofpoising or drug treatment. In other embodiments, the present inventionalso relates to therapeutically active peptides having anti-microbialeffects. In other embodiments, the present invention relates totherapeutically active peptides having the effect of treating cysticfibrosis. In other embodiments, the present invention relates totherapeutically active peptides having the effect of treating orpreventing acute intravascular hemolytic transfusion reactions (AIHTRs).In other embodiments, the present invention relates to therapeuticallyactive peptides having the effect of treating birth asphyxia. Thepresent invention also relates to therapeutically active peptides havingthe effect of treating acute complement-mediated diseases.

As used herein, a peptide sequence is “therapeutically active” if it canbe used for the treatment, remission, or attenuation of a disease state,physiological condition, symptoms or etiological indication(s) orevaluation or diagnosis thereof. A peptide sequence is “prophylacticallyactive” if it can be used to prevent a disease state, physiologicalcondition, symptoms or etiological indications.

The term “subject,” as used herein, means any subject for whomdiagnosis, prognosis, or therapy is desired. For example, a subject canbe a mammal, e.g., a human or non-human primate (such as an ape, monkey,orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse,horse, cattle, or cow.

As used herein, “treat,” “treating,” or “treatment” refers toadministering a therapy in an amount, manner (e.g., schedule ofadministration), and/or mode (e.g., route of administration), effectiveto improve a disorder (e.g., a disorder described herein) or a symptomthereof, or to prevent or slow the progression of a disorder (e.g., adisorder described herein) or a symptom thereof. This can be evidencedby, e.g., an improvement in a parameter associated with a disorder or asymptom thereof, e.g., to a statistically significant degree or to adegree detectable to one skilled in the art. An effective amount,manner, or mode can vary depending on the subject and may be tailored tothe subject. By preventing or slowing progression of a disorder or asymptom thereof, a treatment can prevent or slow deterioration resultingfrom a disorder or a symptom thereof in an affected or diagnosedsubject.

Astrovirus Coat Protein Peptides and Derivatives

CP1 is a peptide derived from human astrovirus coat protein, the peptidecomprising an amino acid sequence of SEQ ID NO: 1.

Using CP1 as the parent peptide, internal deletions of residue 8 toresidue 22 were made for the Δ8-22 peptide (SEQ ID NO: 2) (internaldeletions are shown as dashes in TABLE 1). This peptide was active inall functional assays tested and bound C1q. The 15 amino acid residuesfrom Δ8-22 peptide were scrambled to generate the Polar Assortantpeptide (SEQ ID NO. 3). The scrambled Polar Assortant peptide (SEQ IDNO: 3) is also referred to as PA, PIC1, AstroFend, or AF. As discussedherein, the term “PIC1” peptide includes peptides with the amino acidsequence set forth in SEQ ID NO: 3 as well as peptides with the sameamino acid sequence but with modifications such as PEGylation. The PApeptide was active in all functional assays tested. A series of otherpeptide deletions, substitutions, and modifications of CP1 are describedin U.S. Pat. No. 8,906,845, the contents of which are herebyincorporated by reference in its entirety.

A series of peptide substitutions, and modifications of PA aredisclosed, as shown in TABLES 1, 2, 3, and 4 below. This applicationdiscloses synthetic peptides comprising any one of the amino acidsequences of SEQ ID NOs: 1-47, as shown in TABLES 1-4. As therapeuticagents for humans, the route of administration for the peptide compoundsmay be, for example, topical, enteral, inhaled, parenteral (i.e.intramuscular, subcutaneous, intravenous), or administered through anebulizer, as well as any other routes of administration known in theart.

TABLE 1  Peptide Peptide sequence (N→C) CP1 PAICQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 1) Δ8-22 PAICQRA---------------EIEACI LL (SEQ ID NO: 2)Polar Assortant (also IALILEPICCQERAA known as PA, PIC1, (SEQ ID NO: 3)AstroFend, or AF)Sarcosine Substitutions

Sarcosine (“Sar”) is a natural amino acid, also known asN-methylglycine. Sarcosine is an intermediate in the metabolism ofcholine to glycine. Sarcosine substitution was used to substitute eachresidue of PA sequentially (TABLE 2). Sarcosine substitution can be usedto identify a peptide that retains complement inhibiting activity and issoluble in water.

Disclosed herein are peptide compounds substituted with sarcosine atcertain positions. Table 2 discloses synthetic peptides comprising thesequence of SEQ ID NO: 3, wherein one or more of the amino acids aresubstituted with sarcosine, and wherein the peptides regulate complementactivation and/or have anti-microbial activity. In one or moreembodiments, the amino acids substituted with sarcosine are at positions7, or 9. In one or more embodiments, two or more of the amino acids aresubstituted with sarcosine.

TABLE 2  Peptide Peptide Amino Acid Name Sequence SEQ ID NO PAIALILEPICCQERAA SEQ ID NO: 3 PA-I1Sar (Sar)ALILEPICCQERAA SEQ ID NO: 4PA-A2Sar I(Sar)LILEPICCQERAA SEQ ID NO: 5 PA-L3Sar IA(Sar)ILEPICCQERAASEQ ID NO: 6 PA-I4Sar IAL(Sar)LEPICCQERAA SEQ ID NO: 7 PA-L5SarIALI(Sar)EPICCQERAA SEQ ID NO: 8 PA-E6Sar IALIL(Sar)PICCQERAASEQ ID NO: 9 PA-P7Sar IALILE(Sar)ICCQERAA SEQ ID NO: 10 PA-I8SarIALILEP(Sar)CCQERAA SEQ ID NO: 11 PA-C9Sar IALILEPI(Sar)CQERAASEQ ID NO: 12 PA-C10Sar IALILEPIC(Sar)QERAA SEQ ID NO: 13 PA-Q11SarALILEPICC(Sar)ERAA SEQ ID NO: 14 PA-E12Sar IALILEPICCQ(Sar)RAASEQ ID NO: 15 PA-R13Sar IALILEPICCQE(Sar)AA SEQ ID NO: 16 PA-A14SarIALILEPICCQER(Sar)A SEQ ID NO: 17 PA-A15Sar IALILEPICCQERA(Sar)SEQ ID NO: 18PEGylation

Polyethylene glycol (PEG) is an oligomer or polymer of ethylene oxide.Disclosed herein are peptide compounds that are PEGylated (i.e. have oneor more PEG moieties attached). PEGylation can be used to identify amodified peptide that retains complement inhibiting activity and issoluble in water. One or more PEG moieties can be attached to the Nterminus of the peptide, the C terminus of the peptide, or to both the Nand C terminus of the peptide. The PEGylated peptide compound includesany of the peptides described herein (Tables 1-4) wherein the peptide ismodified through PEGylation of the N terminus, the C terminus, or boththe N terminus and C terminus.

PEGylation was used to attach one or more PEG moieties to the Nterminus, the C terminus, or both the N terminus and C terminus of PA(TABLE 3). In one or more embodiments, 24 PEG moieties are attached tothe N terminus of PA. In one or more embodiments, 24 PEG moieties areattached to the C terminus of PA. In one or more embodiments, 24 PEGmoieties are attached to the N terminus of PA and to the C terminus ofPA. In one or more embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more PEG moieties areattached to the N terminus of PA. In one or more embodiments, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 PEG moieties were attached to the C terminus of PA. In one or moreembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or more PEG moieties are attached to both the Nterminus and the C terminus of PA.

Disclosed herein are PEGylated peptide compounds. This applicationdiscloses synthetic peptides comprising the sequence of SEQ ID NO: 3(PIC1), wherein one or more PEG moieties are attached, and wherein thepeptides regulate complement activation and/or have anti-microbialactivity.

TABLE 3  Peptide  Peptide Amino Acid  Name Sequence SEQ ID NO dPEG24-PA-dPEG24-IALILEPICCQERAA- SEQ ID NO: 19 dPEG24 dPEG24 dPEG24-PAdPEG24-IALILEPICCQERAA SEQ ID NO: 20 PA-dPEG24 IALILEPICCQERAA-dPEG24SEQ ID NO: 21 PA-dPEG20 IALILEPICCQERAA-dPEG20 SEQ ID NO: 22 PA-dPEG16IALILEPICCQERAA-dPEG16 SEQ ID NO: 23 PA-dPEG12 IALILEPICCQERAA-dPEG12SEQ ID NO: 24 PA-dPEG08 IALILEPICCQERAA-dPEG08 SEQ ID NO: 25 PA-dPEG06IALILEPICCQERAA-dPEG06 SEQ ID NO: 26 PA-dPEG04 IALILEPICCQERAA-dPEG04SEQ ID NO: 27 PA-dPEG03 IALILEPICCQERAA-dPEG03 SEQ ID NO: 28 PA-dPEG02IALILEPICCQERAA-dPEG02 SEQ ID NO: 29PEGylation of Peptides in Combination with Amino Acid Substitutions

This application discloses PA/PIC1 that has been modified by acombination of sarcosine substitutions and/or alanine substitutionsand/or PEGylation. A series of peptide substitutions and modificationsof PA are disclosed, as shown in TABLE 4 below. The modified andsubstituted peptides can be used to identify a peptide that retainscomplement inhibiting activity and is soluble in water. This applicationdiscloses a synthetic peptide comprising the sequence of SEQ ID NO: 3,wherein one or more of the amino acids are substituted with sarcosine oralanine, wherein the peptides regulate complement activation and/or hasanti-microbial activity. This application also discloses a syntheticpeptide comprising the sequence of SEQ ID NO: 3, wherein one or more ofthe amino acids are substituted with sarcosine or alanine and one ormore PEG moieties is attached to the N terminus of the peptides, the Cterminus of the peptides, or to both the N and C terminus of thepeptides, wherein the peptides regulate complement activation and/orhave anti-microbial activity.

In one or more embodiments, two or more of the amino acids aresubstituted with sarcosine. In one or more embodiments, two or more ofthe amino acids are substituted with alanine. In one or moreembodiments, one or more of the amino acids are substituted withsarcosine and one or more of the amino acids are substituted withalanine. The substituted peptides can further be modified by PEGylationas described above.

TABLE 4  Peptide Name Peptide Amino  and Controls Acid SequenceSEQ ID NO Water — DMSO — PA-dPEG24 IALILEPICCQERAA-dPEG24 SEQ ID NO: 21PA-C9SarC10A IALILEPI(Sar) A QERAA SEQ ID NO: 30 PA-C9SarD10IALILEPI(Sar)QERAA SEQ ID NO: 31 PA-P7SarC9Sar IALILE(Sar)I(Sar)CQERAASEQ ID NO: 32 PA-E6Sar- IALIL(Sar)PICCQERAA- SEQ ID NO: 33 dPEG24 dPEG24PA-Q11Sar- IALILEPICC(Sar)ERAA- SEQ ID NO:34 dPEG24 dPEG24 PA-R13Sar-IALILEPICCQE(Sar)AA- SEQ ID NO:35 dPEG24 dPEG24 PA-A14Sar-IALILEPICCQER(Sar)A- SEQ ID NO:36 dPEG24 dPEG24 E6SarP7SarIALIL(Sar)(Sar)ICCQERAA SEQ ID NO: 37 E6SarC9Sar IALIL(Sar)PI(Sar)CQERAASEQ ID NO: 38 Q11SarP7Sar IALILE(Sar)ICC(Sar)ERAA SEQ ID NO: 39Q11SarC9Sar IALILEPI(Sar)C(Sar)ERAA SEQ ID NO: 40 R13SarP7SarIALILE(Sar)ICCQE(Sar)AA SEQ ID NO: 41 R13SarC9SarIALILEPI(Sar)CQE(Sar)AA SEQ ID NO: 42 A14SarP7SarIALILE(Sar)ICCQER(Sar)A SEQ ID NO: 43 A14SarC9SarIALILEPI(Sar)CQER(Sar)A SEQ ID NO: 44 E6AE12A- IALIL A PICCQ ARAA-dPEG24 SEQ ID NO: 45 dPEG24 E6AE12AC9Sar IALIL A PI(Sar)CQ A RAASEQ ID NO: 46 E6AE12AP7Sar IALIL A (Sar)ICCQ A RAA SEQ ID NO: 47Antimicrobial Activity of PIC1 Peptides

There is currently a critical need for new antibiotics as manymicroorganisms have become resistant to currently prescribedantibiotics. Additionally, most antibiotics are derived from othermicrobial organisms that bacteria have competed against for space andenergy over the millennia, leading to rapid and predictable emergence ofresistance.

The CP1 peptide was initially identified by its weak homology to thehuman neutrophil defensing peptide 1 (HNP-1). In addition to inhibitingcomplement activation, HNP-1 has the ability to inhibit bacterialgrowth. The PIC1peptides described herein, including SEQ ID NOs: 3-47,are very different in amino acid sequence to HNP-1 and have no knownhomologs in nature. Surprisingly, in some aspects, the PIC1 peptideshave anti-bacterial activity.

In some aspects, the disclosed peptide compounds have a directantimicrobial effect and are, thus, ideal for inhibiting the growth ofbacterial diseases. The disclosed peptide compounds can be used toprevent and treat diseases mediated by bacteria. In some embodiments,the disclosed peptide compounds can be used to prevent and treat Grampositive and Gram negative bacterial infections. In some embodiments,the disclosed peptide compounds can be used to prevent and treat, forexample, Pseudomonas aeruginosa, MRSA, and carbapenemase-resistantenterobacteriacea (CREs) (e.g. resistant Klebsiella pneumonia). Thedisclosed peptide compounds can be used to prevent and treat bacterialvaginosis and vaginitis. In one or more embodiments, the disclosedpeptide compounds kill the causative organism of bacterial vaginosis andalso blocks the inflammation, which disrupts barrier defenses increasingrisk of HIV transmission. As the PIC1 peptides have no homology withknown proteins or peptides, this has the potential to decrease thelikelihood of emergence of resistance.

The disclosed peptide compounds can also enhance growth ofLactobacillus. In some embodiments of the invention, L. acidophilus andL. leichmannii are enhanced by the disclosed peptide compounds. Incertain aspects, the invention provides a method of enhancingLactobacillus growth by administering a therapeutically effective amountof at least one synthetic peptide selected from the group consisting ofSEQ ID NOS: 3-47.

Bacterial vaginosis and vaginitis are currently treated with systemicantibiotics. The disclosed peptide compounds can be used to prevent andtreat bacterial vaginosis and vaginitis by local administration (e.g.,topical administration) or by systemic administration (e.g., intravenousadministration).

In certain aspects, the invention provides a method of treating abacterial infection comprising administering to a subject apharmaceutical composition comprising a therapeutically effective amountof a synthetic peptide comprising the amino acid sequence andmodifications selected from SEQ ID NOs: 3-47. In one or moreembodiments, the bacteria are Staphylococcus aureus, Klebsiellapneumonia, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydiatrachomatis, or Gardnerella sp. In one or more embodiments, the subjecthas cystic fibrosis, and the peptide compounds treat cystic fibrosis. Inone or more embodiments, the subject has gonorrhea or chlamydia, and thepeptide compounds treat gonorrhea or chlamydia. In one or moreembodiments, the subject has pneumonia, and the peptide compounds treatpneumonia.

In certain aspects, the invention provides a method of treatingbacterial vaginitis comprising administering to a subject apharmaceutical composition comprising a therapeutically effective amountof a synthetic peptide comprising the amino acid sequence andmodifications selected from SEQ ID NOs: 3-47.

The Complement System and Diseases Associated with its Dysregulation

While complement is a vital host defense against microorganisms such asbacteria and some enveloped viruses, its unchecked activation can causedevastating host cell damage. Host tissue damage mediated by complementhas been implicated in a wide variety of diseases, including autoimmunepathologies such as: rheumatoid arthritis, systemic lupus erythematosus,multiple sclerosis, myasthenia gravis, autoimmune hemolytic anemia,membranoproliferative glomerulonephritis, and serum sickness. It hasalso been identified as contributing to the development of the followingdiseases: Adult Respiratory Distress Syndrome (ARDS),ischemia-reperfusion injuries (including stroke and myocardialinfarction), allo- and xeno-transplantation complications (includinghyperacute rejection and graft versus host disease (GVHD), Alzheimer'sdisease, burn injuries, hemodialysis damage, cardiopulmonary bypassdamage, and paroxysmal nocturnal hemoglobinuria (PNH).

Hereditary angioedema (HAE) is a rare genetic disorder caused by reducedlevels of or non-functional C1-inhibitor; symptoms of HAE include acuteedema. C1-inhibitor naturally regulates C1 activation, and treatment ofacute edema requires substantial infusion of C1-inhibitor or plasmatransfusion. Because astrovirus CP functionally blocks C1 activation,using the disclosed peptide compounds to treat HAE fulfills atherapeutic need because C1-inhibitor has to be purified from human serafrom multiple subjects and, therefore, could be contaminated with humanblood-borne pathogens. Therapeutic administration of the disclosedpeptide compounds regulates C1 either in adjunct therapy withC1-inhibitor or as a stand-alone therapeutic treatment.

The disclosed peptide compounds can selectively regulate C1q and MBLactivation without affecting alternative pathway activity and are, thus,ideal for preventing and treating diseases mediated by the dysregulatedactivation of the classical and lectin pathways. Specific blockade ofclassical and lectin pathways are particularly needed, as both of thesepathways have been implicated in ischemia-reperfusion induced injury inmany animal models. [Castellano et al., “Therapeutic targeting ofclassical and lectin pathways of complement protects fromischemia-reperfusion-induced renal damage.” Am J Pathol. 2010;176(4):1648-59; Lee et al., “Early complement factors in the localtissue immunocomplex generated during intestinal ischemia/reperfusioninjury.” Mol. Immunol. 2010 February; 47(5): 972-81; Tjernberg, et al.,“Acute antibody-mediated complement activation mediates lysis ofpancreatic islets cells and may cause tissue loss in clinical islettransplantation.” Transplantation. 2008; April 27; 85(8):1193-9; Zhanget al. “The role of natural IgM in myocardial ischemia-reperfusioninjury.” J Mol Cell Cardiol. 2006 July; 41(1):62-7). The alternativepathway is essential for immune surveillance against invading pathogens,and humans with alternative pathway defects suffer severe bacterialinfections. By binding and inactivating C1q and MBL, the peptidecompounds can efficiently regulate classical and lectin pathwayactivation while leaving the alternative pathway intact.

The term “regulate,” as used herein, refers to i) controlling, reducing,inhibiting or regulating the biological function of an enzyme, protein,peptide, factor, byproduct, or derivative thereof, either individuallyor in complexes; ii) reducing the quantity of a biological protein,peptide, or derivative thereof, either in vivo or in vitro; or iii)interrupting a biological chain of events, cascade, or pathway known tocomprise a related series of biological or chemical reactions. The term“regulate” may thus be used, for example, to describe reducing thequantity of a single component of the complement cascade compared to acontrol sample, reducing the rate or total amount of formation of acomponent or complex of components, or reducing the overall activity ofa complex process or series of biological reactions, leading to suchoutcomes as cell lysis, formation of convertase enzymes, formation ofcomplement-derived membrane attack complexes, inflammation, orinflammatory disease. In an in vitro assay, the term “regulate” mayrefer to the measurable change or reduction of some biological orchemical event, but the person of ordinary skill in the art willappreciate that the measurable change or reduction need not be total tobe “regulatory.”

In one or more aspects, the disclosed peptide compounds can be used totreat an inflammatory disease. In one or more embodiments, the disclosedpeptide compounds can be used to treat an autoimmune disease. In one ormore embodiments, the disclosed peptide compounds can be used to treathemolytic transfusion reactions, cold-agglutinin disease, immune-complexdiseases (e.g. serum sickness), thalassemia, sickle cell disease, ABOincompatibility (e.g. neonatal jaundice), acute/hyperacute solid organtransplantation rejection, solid organ transplantation warm/coldischemia, instant blood-mediated inflammatory reaction (IBMIR), systemiclupus erythematosus (SLE), rheumatoid arthritis, ischemia-reperfusioninjury, myocardial infarct, stroke, hypoxic ischemic encephalopathy(e.g. birth asphyxia brain injury), traumatic brain injury, coronaryartery bypass surgery, cystic fibrosis, wound healing, cancer,Alzheimer's disease, Parkinson's disease, paroxysmal nocturnalhemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma,chronic obstructive pulmonary disease (COPD), Crohn's disease, Sepsissyndrome/ARDS/SIRS, glomerulonephritis (e.g. lupus nephritis,anti-glomerular basement membrane disease, anti-neutrophil cytoplasmicautoantibody-induced, membranoproliferative glomerulonephritis (e.g.dense deposit disease), membranous nephropathy), IgA nephropathy, or C3glomerulopathy.

Complement-Mediated Acute Intravascular Hemolytic Transfusion Reaction(AIHTR)

Acute intravascular hemolytic transfusion reaction (“ATR,” “AHTR,” or“AIHTR”) is a type of transfusion reaction that is associated withhemolysis. AIHTR can result in complement mediated lysis and rapiddestruction of donor red blood cells. AIHTRs have a broad clinicalpresentation from mild and transitory signs and symptoms to seriouscases of shock, disseminated intravascular coagulation, renal failureand death.

The American Red Cross estimates that 30 million blood components aretransfused every year in the United States (US) [Barbee I. Whitaker P SH, PhD, The 2011 National Blood Collection and Utilization SurveyReport; 2011]. Approximately 70,000 US patients living with sickle celldisease and 1.6 million individuals diagnosed with cancer require bloodtransfusions routinely as part of their disease management. Bloodtransfusions are life-saving, but carry the risk of a variety ofreactions some of which are potentially life-threatening such as acuteintravascular hemolytic transfusion reactions (AIHTRs) (Murphy M F,Waters J H, Wood E M, Yazer M H. “Transfusing blood safely andappropriately.” BMJ. 2013; 347:f4303]. AIHTR is estimated to occur inone-fifth of total transfusions (Refaai M A, Blumberg N. “Thetransfusion dilemma—weighing the known and newly proposed risks of bloodtransfusions against the uncertain benefits.” Best practice & researchClinical anaesthesiology. 2013; 27(1): 17-35). Individuals receivingfrequent blood transfusions will develop alloantibodies andautoantibodies to red blood cell (RBC) antigens over time makingcross-matching increasingly difficult and thus increasing the risk ofAIHTR (Aygun B, Padmanabhan S, Paley C, Chandrasekaran V. “Clinicalsignificance of RBC alloantibodies and autoantibodies in sickle cellpatients who received transfusions.” Transfusion. 2002; 42(1):37-43].Current transfusion safe-guards include ‘typing’, ‘antibody screening’as well as ‘cross matching’, and while these measures have madetransfusions safer than ever before, transfusion reactions still occur(Osterman J L, Arora S. “Blood product transfusions and reactions.”Emergency medicine clinics of North America. 2014; 32(3):727-738]. AIHTRoccurs when host antibodies bind to the transfused erythrocytesinitiating classical complement pathway activation leading to thegeneration of the inflammatory mediators C3a and C5a as well as C3bopsonization and hemolysis of the transfused cells via the membraneattack complex (MAC) (Stowell S R, Winkler A M, Maier C L, et al.“Initiation and regulation of complement during hemolytic transfusionreactions.” Clinical & developmental immunology. 2012; 2012:307093].While the role of complement in AIHTR is well recognized, to date, onlyone case has been reported describing clinical intervention of an AIHTRby inhibiting generation of the complement anaphylatoxins C3a and C5a[Weinstock C, Mohle R, Dorn C, et al. “Successful use of eculizumab fortreatment of an acute hemolytic reaction after ABO-incompatible redblood cell transfusion.” Transfusion. 2015; 55(3):605-610].

The classical pathway of complement acts as an amplification cascadeafter initial activation of complement complex C1 by antibodies (Frank MM A J ed Complement system. In: Austen K F, Atkinson J P, Cantor H I ed.Samter's Immunologic Disease. New York: Lippincott Williams and Wilkins;2001]. The C1 complex consists of the pattern recognition molecule C1qand the serine protease tetramer C1r-C1 s-C1 s-C1r. Upon binding of C1qto IgM or clustered IgG antibodies, C1q undergoes a conformationalchange to activate C1r-C1 s-C1 s-C1r and initiate classicalpathway-mediated complement activation. The Peptide Inhibitor ofComplement C1 (PIC1) described herein is a peptide inhibitor of theclassical pathway of complement (Mauriello C T, Pallera H K, Sharp J A,et al. “A novel peptide inhibitor of classical and lectin complementactivation including ABO incompatibility.” Mol Immunol. 2013;53(1-2):132-139; Sharp J A, Whitley P H, Cunnion K M, Krishna N K.“Peptide inhibitor of complement C1, a novel suppressor of classicalpathway activation: mechanistic studies and clinical potential.”FrontImmunol. 2014; 5:406; Gronemus J Q, Hair P S, Crawford K B,Nyalwidhe J O, Cunnion K M, Krishna N K. “Potent inhibition of theclassical pathway of complement by a novel C1q-binding peptide derivedfrom the human astrovirus coat protein.” Mol Immunol. 2010;48(1-3):305-313). PIC1 inhibits antibody-initiated activation of C1 bybinding C1q and preventing activation of C1r-C1 s-C1 s-C1r12. PIC1 alsohas also been shown to inhibit classical complement pathway-mediated ABOincompatible hemolysis of human erythrocytes in vitro (Mauriello C T,Pallera H K, Sharp J A, et al. “A novel peptide inhibitor of classicaland lectin complement activation including ABO incompatibility.” MolImmunol. 2013; 53(1-2):132-139; Sharp J A, Whitley P H, Cunnion K M,Krishna N K. “Peptide inhibitor of complement C1, a novel suppressor ofclassical pathway activation: mechanistic studies and clinicalpotential.” FrontImmunol2014; 5:406; Shah T A, Mauriello C T, Hair P S,et al. “Complement inhibition significantly decreases red blood celllysis in a rat model of acute intravascular hemolysis.” Transfusion.2014). When the aqueous polyethylene glycol (PEG)-conjugated version ofthe PIC1, PA-dPEG24 (SEQ ID NO: 21), described herein is administeredintravascularly into rats, it can achieve greater than 90% systemicinhibition of the animal's serum complement levels within 30 seconds(Sharp J A, Whitley P H, Cunnion K M, Krishna N K. “Peptide inhibitor ofcomplement C1, a novel suppressor of classical pathway activation:mechanistic studies and clinical potential.” Front Immunol. 2014;5:406]. The ability of PIC1 to inhibit complement activation makes it anideal molecule to block the rapid complement-mediated hemolysis thattypifies AIHTR.

A rat AIHTR disease model based on xenotransfusion of human RBCs wasestablished (Shah T A, Mauriello C T, Hair P S, et al. “Complementinhibition significantly decreases red blood cell lysis in a rat modelof acute intravascular hemolysis.” Transfusion. 2014]. Wistar rats havenatural hemagglutinins in their serum that will bind to human ABerythrocytes (Shah et al; Aptekman P M, Bogden A E. “Characterization ofthe natural hemagglutinins in normal rat serum associated with anegative phase following tumor implantation.” Cancer Research. 1956;16(3):216-221). Upon transfusion of human erythrocytes, they willproduce a rapid ABO incompatibility-like hemolysis viaantibody-initiated classical complement pathway activation (YazdanbakhshK, Kang S, Tamasauskas D, Sung D, Scaradavou A. “Complement receptor 1inhibitors for prevention of immune-mediated red cell destruction:potential use in transfusion therapy.” Blood. 2003; 101(12):5046-5052].

PIC1 peptides, including PA (SEQ ID NO: 3) and PA-dPEG24 (SEQ ID NO:21), can block complement-mediated lysis of AB human red blood cells(RBC) by O serum in vitro. This assay mimics ABO incompatibility.PA-dPEG24 has efficacy in a rodent model of AIHTR demonstratinginhibition of human RBC lysis in a prevention and rescue scenario. ThusPIC1 peptides can be used in the treatment of transfusion reactions inhumans for which no current therapy exists.

Current blood banking organization or transfusion medicine practice doesnot have a method to directly evaluate risk for complement mediated RBClysis between donor and recipient. The disclosed peptide compounds canbe used as a diagnostic tool. The disclosed peptide compounds can alsobe used as a prophylactic treatment to prevent AIHTR or as a rescuetreatment during an AIHTR has significant clinical implications. Thedisclosed peptide compounds can prevent the high concentrations of freehemoglobin that cause acute kidney injury in AIHTR. There are currentlyno therapies for ATRs other than supportive care.

The disclosed peptide compounds can be used to detectcomplement-mediated lysis of RBCs and are thus ideal for discriminatingif an erythrocyte transfusion may cause AIHTR. The disclosed peptidecompounds can be used to predict AIHTR.

In certain aspects, the invention provides a method of treating acutetransfusion reaction comprising administering to a subject apharmaceutical composition comprising a therapeutically effective amountof a synthetic peptide comprising the amino acid sequence andmodifications selected from SEQ ID NOs: 3-47. In one or moreembodiments, the pharmaceutical composition is administered before thesubject is administered a blood transfusion. In one or more embodiments,the pharmaceutical composition is administered after the subject isadministered a blood transfusion. In one or more embodiments, thepharmaceutical composition is administered before and after the subjectis administered a blood transfusion.

Complement Effectors in Cystic Fibrosis (CF)

In cystic fibrosis (CF), lung damage is believed to be mediated by acycle of obstruction, infection, and inflammation. It has beendetermined that complement effectors are elevated in the lung fluids ofCF patients compared to normal controls.

Cystic fibrosis (CF) afflicts 30,000 individuals in the United States(Boyle M P. “Adult cystic fibrosis.” JAMA 2007; 298(15):1787-93) withrespiratory failure causing the majority of deaths. Progressivedestruction of lung parenchyma is mediated by a cycle of obstruction,infection with bacterial pathogens, and inflammation (Rowe S M, MillerS, Sorscher E J, “Cystic fibrosis.” N Engl J Med 2005;352(19):1992-2001). As the cycle repeats there is progression from lungdamage to lung scarring and finally pulmonary failure (Gibson R L, BurnsJ L, Ramsey B W. “Pathophysiology and management of pulmonary infectionsin cystic fibrosis.” Am J Respir Crit Care Med 2003; 168(8):918-51].

The most destructive inflammatory cascade in the human body is thecomplement system, which contributes to host tissue damage in numerousinflammatory disease processes (Ricklin D, Lambris J D.“Complement-targeted therapeutics.” Nat Biotechnol 2007;25(11):1265-75). Recent evidence has shown that complement proteins aremajor constituents of lung fluid in CF patients and normal humans, whereC3 and C4 account for two of the four most prevalent proteins (Gharib SA, Vaisar T, Aitken M L, et al. “Mapping the lung proteome in cysticfibrosis.” J Proteome Res 2009; 8(6):3020-8). This suggests thatcomplement may play a much larger role in CF lung inflammation than whathas been previously suspected. Antibody binding to bacteria can activatethe classical complement pathway via the initiating component C1 (FIG.9). C4 is a cascade component for the classical (i.e. antibodyinitiated) pathway leading to formation of the opsonin C4b anddownstream activation of C3 (Lambris J D, Sahu A, Wetsel R A. “Thechemistry and biology of C3, C4, and C5.” In: Volanakis J E, Frank M M,(eds). The human complement system in health and disease. New York:Marcel Dekker; 1998, 83-118.). C3 is the central complement component,which upon activation generates the complement effector C3a andcovalently binds cells with the opsonic fragments C3b and iC3b. C3b theninitiates activation of C5, generating the extremely potentanaphylatoxin CSa. C5a is among the most powerful stimulants forneutrophil migration and activation, leading to oxidative burst anddegranulation (Lambris J D, Sahu A, Wetsel R A. “The chemistry andbiology of C3, C4, and C5.” In: Volanakis J E, Frank M M, (eds). Thehuman complement system in health and disease. New York: Marcel Dekker;1998, 83-118; Tralau T, Meyer-Hoffert U, Schroder J M, et al. “Humanleukocyte elastase and cathepsin G are specific inhibitors ofC5a-dependent neutrophil enzyme release and chemotaxis.” Exp Dermatol2004; 13(5):316-25). Neutrophil death, which follows degranulation, is amajor source of the viscous DNA that contributes to the obstruction ofairways in the CF lung (Dwyer M, Shan Q, D'Ortona S, et al. “CysticFibrosis Sputum DNA Has N ETosis Characteristics and NeutrophilExtracellular Trap Release Is Regulated by MacrophageMigration-Inhibitory Factor.” J Innate Immun 2014; Hodson M E.“Aerosolized dornase alfa (rhDNase) for therapy of cystic fibrosis.” AmJ Respir Crit Care Med 1995; 151(3 Pt 2):570-4]. Among the neutrophilgranule products released is neutrophil elastase, which is a majorcontributor to parenchymal lung damage in CF (Gifford A M, Chalmers J D.“The role of neutrophils in cystic fibrosis.” Curr Opin Hematol 2014;21(1):16-22; Le Gars M, Descamps D, Roussel D, et al. “Neutrophilelastase degrades cystic fibrosis transmembrane conductance regulatorvia calpains and disables channel function in vitro and in vivo.” Am JRespir Crit Care Med 2013; 187(2):170-9; Sagel S D, Wagner B D, AnthonyM M, et al. “Sputum biomarkers of inflammation and lung function declinein children with cystic fibrosis.” Am J Respir Crit Care Med 2012;186(9):857-65]. Thus, complement activation may play a significant rolein neutrophil recruitment and activation in CF lungs, contributing totissue damage. Additional properties of C5a that may also contribute toCF lung disease are stimulation of histamine release, enhancement ofvascular permeability, and inducement of smooth muscle contraction(Lambris J D, Sahu A, Wetsel R A. “The chemistry and biology of C3, C4,and C5.” In: Volanakis J E, Frank M M, (eds). The human complementsystem in health and disease. New York: Marcel Dekker; 1998, 83-11]. Theknown inflammatory properties of C5a are consistent with the increasingevidence of the role of C5a in inflammatory lung diseases (Schmudde I,Strover H A, Vollbrandt T, et al. “C5a receptor signalling in dendriticcells controls the development of maladaptive Th2 and Th17 immunity inexperimental allergic asthma.” Mucosal Immunol 2013; 6(4):807-25;Bosmann M, Ward P A. “Role of C3, C5 and anaphylatoxin receptors inacute lung injury and in sepsis.” Adv Exp Med Biol 2012; 946:147-59],including acute lung injury. Thus, although not bound by any theory,multiple lines of reasoning suggest that complement-mediatedinflammation may be a major contributor to inflammatory lung damage inCF.

Some investigation into the potentially important role of C5a in the CFlung has been performed. In 1986, Fick et al. described the presence ofincreased amounts of C5a, measured by radioimmunoassay in thebronchoalveolar lavage (BAL) of nine CF patients with clinically stablelung disease compared with BAL from healthy controls. (Fick R B, Jr.,Robbins R A, Squier S U, et al. “Complement activation in cysticfibrosis respiratory fluids: in vivo and in vitro generation of C5a andchemotactic activity.” Pediatr Res 1986; 20(12):1258-68). The CF BALfluids were chemotactic for neutrophils, which appeared to correlatewith C5a concentrations. The BAL fluids showed evidence of priorcomplement activation by the presence of C3c, assayed bycrossed-immunoelectrophoresis. Two CF patients with the lowest C5ameasurements were noted to have normal FEV₁ and FVC measurements,suggesting a potential association with lung damage. However, no furtherstudies were then performed to test whether C5a concentrations in CFlung fluid correlate with either acute lung exacerbations or chroniclung disease progression in CF.

Although not bound by any mechanism, the disclosed peptide compounds canselectively regulate C1q and MBL activation without affectingalternative pathway activity and are, thus, ideal for treating cysticfibrosis. In one or more embodiments, the disclosed peptide compoundscan be used to treat cystic fibrosis. In one or more embodiments, thedisclosed peptide compounds can be used as anti-inflammatory andanti-microbial agents. In one or more embodiments, the disclosed peptidecompounds can be used to treat cystic fibrosis by administrating thepeptide(s) via nebulization directly into the lung. In one or moreembodiments, administration of the disclosed peptide compound(s) vianebulization mitigates lung destruction caused by cystic fibrosis. Thedisclosed peptide compounds have clinical benefit for CF patients byslowing the progression of lung damage leading to increased life spanand quality of life.

In certain aspects, the invention provides a method of treating cysticfibrosis comprising administering to a subject a pharmaceuticalcomposition comprising a therapeutically effective amount of a syntheticpeptide comprising an amino acid sequence and modifications selectedfrom SEQ ID NOs: 3-47.

Modulation of C1q Interaction with C1q Receptors

C1q interactions with C1q receptors appear to play important roles inhomeostatic functions such as scavenging of apoptotic cellular debrisand immune complexes as well as T cell signaling through antigenpresenting cells (macrophage and dendritic cells). Currently, noclinical pharmacological agents modulate the interaction of C1q with C1qreceptors.

The disclosed peptide compounds can be used to block C1q binding to C1qreceptors, including calreticulin/cC1qR. The ability of the disclosedpeptide compounds to block binding of C1q to cellular receptors may havean important role in modulating intracellular signaling processesmediated by C1q binding to C1q receptors. The disclosed peptidecompounds can be used to regulate the complement response in diseaseprocesses such as systemic lupus erythematosus and cancer.

Brain Damage in Birth Asphyxia

Complement activation is instrumental in the development ofischemia-reperfusion injuries such as neonatal hypoxic ischemicencephalopathy (HIE). Therapeutic hypothermia (HT) offers only an 11%reduction in death or disability. Published in vitro data has shown thatHT paradoxically increase pro-inflammatory complement activationpotentially limiting its benefit.

PIC1 peptides can reduce brain infarct volumes without prolongedsystemic complement depletion. PIC1 peptides can be a useful adjunct toHT to improve neurological outcomes in HIE.

The disclosed peptide compounds can be us to prevent brain damage frombirth asphyxia. The disclosed peptide compounds can also be used toprevent ischemia-reperfusion injury (IRI) in diseases such as myocardialinfarct, coronary artery bypass surgery, stroke, etc. The disclosedpeptide compounds can also be used to prevent hyper-acute and acutesolid organ transplantation rejection, which are classical complementpathway mediated events.

In certain aspects, the invention provides a method of treating birthasphyxia comprising administering to a subject a pharmaceuticalcomposition comprising a therapeutically effective amount of a syntheticpeptide comprising an amino acid sequence and modifications selectedfrom SEQ ID NOs: 3-47. In some embodiments, the subject is furthertreated by therapeutic hypothermia.

In certain aspects, the invention provides a method of treating hypoxicischemic encephalopathy comprising administering to a subject apharmaceutical composition comprising a therapeutically effective amountof a synthetic peptide comprising an amino acid sequence andmodifications selected from SEQ ID NOs: 3-47. In some embodiments, thesubject is further treated by therapeutic hypothermia.

Myeloperoxidase (MPO) Activity

Myeloperoxidase (MPO) is an enzyme from neutrophils that createshypochlorite (bleach) in acute inflammation and damages invading andhost cells alike. This enzyme is known to be destructive to host tissuesin Cystic Fibrosis (CF) and Hypoxic Ischemic Encephalopathy (HIE).

In some embodiments, PIC1 blocked the enzymatic activity of MPO in thesputum of cystic fibrosis patients. In some embodiments, PIC1 blockedenzymatic activity in brain tissue from HIE patents. In someembodiments, MPO activity present in the lysates of purified humanneutrophils can be directly inhibited by PIC1. In some embodiments, theinvention demonstrates that PIC1 has anti-inflammatory activity againstcystic fibrosis and HIE.

Autoimmune Hemolytic Anemia

The present disclosure provides peptide compounds capable of treatingautoimmune hemolytic anemia. Autoimmune hemolytic anemia is a group ofdisorders characterized by a malfunction of the immune system thatproduces autoantibodies, which attack red blood cells as if they weresubstances foreign to the body. Autoimmune hemolytic anemia may becharacterized by one or more of elevated serum bilirubin, excess urinaryurobilinogen, reduced plasma haptoglobin, raised serum lacticdehydrogenase (LDH), hemosiderinuria, methemalbuminemia, spherocytosis,reticulocytosis, and/or erythroid hyperplasia of the bone marrow. Incertain aspects of the invention, treating autoimmune hemolytic anemiacomprises administering a peptide compounds selected from the groupconsisting of SEQ ID NO: 3-47.

Pharmaceutical Formulation and Administration

The present disclosure provides pharmaceutical compositions capable ofregulating the complement system, comprising at least one peptidecompound, as discussed above, and at least one pharmaceuticallyacceptable carrier, diluent, stabilizer, or excipient. Pharmaceuticallyacceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed. They can besolid, semi-solid, or liquid. The pharmaceutical compositions of thepresent invention can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,or syrups.

Some examples of pharmaceutically acceptable carriers, diluents,stabilizers, or excipients include: lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The pharmaceutical compositions of the present invention canbe formulated using procedures known in the art to provide quick,normal, or sustained or delayed release of the active ingredient.

The disclosure relates to a method of regulating the complement systemin a subject comprising administering to a subject the compositionsdescribed above. The pharmaceutical compositions of the presentinvention are prepared by mixing the peptide compound having theappropriate degree of purity with pharmaceutically acceptable carriers,diluents, or excipients. Examples of formulations and methods forpreparing such formulations are well known in the art. Thepharmaceutical compositions of the present invention are useful as aprophylactic and therapeutic agent for various disorders and diseases,as set forth above. In one embodiment, the composition comprises atherapeutically effective amount of the peptide compound. In anotherembodiment, the composition comprises at least one other activeingredient effective in treating at least one disease associated withcomplement-mediated tissue damage. The term “therapeutically effectiveamount,” as used herein, refers to the total amount of each activecomponent that is sufficient to show a benefit to the subject.

The therapeutically effective amount of the peptide compound variesdepending on several factors, such as the condition being treated, theseverity of the condition, the time of administration, the route ofadministration, the rate of excretion of the compound employed, theduration of treatment, the co-therapy involved, and the age, gender,weight, and condition of the subject, etc. One of ordinary skill in theart can determine the therapeutically effective amount. Accordingly, oneof ordinary skill in the art may need to titer the dosage and modify theroute of administration to obtain the maximal therapeutic effect.

The effective daily dose generally is within the range of from about0.001 to about 200 milligrams per kilogram (mg/kg) of body weight,preferably about 80 to about 160 mg/kg, more preferably about 0.1 toabout 20 mg/kg. This dose can be achieved through a 1-6 time(s) dailydosing regimen. Alternatively, optimal treatment can be achieved througha sustained release formulation with a less frequent dosing regimen.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example, by the oral, nasal, topical (includingbuccal, sublingual, or transdermal), or parenteral (includingsubcutaneous, intracutaneous, intramuscular, intraarticular,intraperitoneal, intrasynovial, intrasternal, intrathecal,intralesional, intravenous, or intradermal injections or infusions)route. For human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity standards, asrequired by the offices of the Food and Drug Administration (FDA).

Combination Therapies

A further embodiment of the invention provides a method of preventing ortreating a disease associated with complement-mediated tissue damage,comprising administering to a subject a pharmaceutical composition ofthe present invention. While the pharmaceutical compositions of thepresent invention can be administered as the sole active pharmaceuticalagent, they can also be used in combination with one or more therapeuticor prophylactic agent(s) that is(are) effective for preventing ortreating the disease. In this aspect, the method of the presentinvention comprises administrating a pharmaceutical composition of thepresent invention before, concurrently, and/or after one or moreadditional therapeutic or prophylactic agents effective in treating atleast one disease associated with complement-mediated tissue damage.

For example, the pharmaceutical compositions of the present inventioncan be used to treat brain asphyxia or hypoxic ischemic encephalopathy,either alone or in combination with therapeutic hypothermia.

For example, the pharmaceutical compositions of the present inventioncan be used to treat rheumatoid arthritis, either alone or incombination with a non-steroidal anti-inflammatory agent (NSAID), acorticosteroid, or a disease modifying anti-rheumatic drug (DMARD).

Examples of NSAIDs include: salicylates (such as aspirin, amoxiprin,benorilate, choline magnesium salicylate, diflunisal, faislamine, methylsalicylate, magnesium salicylate, and salicyl salicylate (salsalate)),arylalkanoic acids (such as diclofenac, aceclofenac, acemetacin,bromfenac, etodolac, indometacin, ketorolac, nabumetone, sulindac, andtolmeti), 2-arylpropionic acids (such as ibuprofen, carprofen, fenbufen,fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, tiaprofenicacid, and suprofen), N-arylanthranilic acids (such as mefenamic acid andmeclofenamic acid), pyrazolidine derivatives (such as phenylbutazone,azapropazone, metamizole, oxyphenbutazone, and sulfinprazone), oxicams(such as piroxicam, lornoxicam, meloxicam, and tenoxicam), COX-2inhibitors (such as etoricoxib, lumiracoxib, and parecoxib),sulphonanilides such as nimesulide, and others such as licofelone andomega-3 fatty acids.

Examples of corticosteroids include: triamcinolone (Aristocort®),cortisone (Cortone® Acetate Tablets), dexamethasone (Decadron® Elixir),prednisone (Deltasone®), and methylprednisolone (Medrol®).

Examples of DMARDs include: methotrexate (Rheumatrex®), leflunomide(Arava®), etanercept (Enbrel®), infliximab (Remicade®), adalimumab(Humira®), anakinra (Kineret®), sulfasalazine (Azulfidine EN-Tabs®),antimalarials, gold salts, d-penicillamine, cyclosporin A,cyclophosphamide and azathioprine.

Soliris (eculizumab) is a humanized anti-CS monoclonal antibody. It hasbeen approved by the FDA for the treatment of the rare form of hemolyticanemia, paroxysmal nocturnal hemoglobinuria. In one embodiment, thepharmaceutical compositions of the present invention can be used incombination with Soliris in treating paroxysmal nocturnalhemoglobinuria, atypical hemolytic uremic syndrome, heart disease,pulmonary diseases, autoimmune diseases, asthma, as well as theancillary care of transplants.

The pharmaceutical compositions of the present invention can beadministered with additional agent(s) in combination therapy, eitherjointly or separately, or by combining the pharmaceutical compositionsand the additional agent(s) into one composition. The dosage isadministered and adjusted to achieve maximal management of theconditions. For example, both the pharmaceutical compositions and theadditional agent(s) are usually present at dosage levels of betweenabout 10% and about 150%, more preferably, between about 10% and about80%, of the dosage normally administered in a mono-therapy regimen.

EXAMPLES

The invention is further illustrated by the following examples, providedfor illustrative purposes only. They are not to be construed as limitingthe scope or content of the invention in any way.

Example 1: Solubility and Hemolytic Assay of Sarcosine (SAR)Substitution Peptides

Methods: Hemolytic Assay.

Peptides were diluted to 0.77 mM in factor B-depleted human sera(Complement Technologies, Inc.) and incubated for 1 hour at 37° C. Thesepeptides were then diluted with GVBS⁺⁺ to equal 2.5% serum, of which0.25 ml was combined with 0.4 ml of GVBS⁺⁺ and 0.1 ml of sensitizedsheep red blood cells (RBCs) and again incubated for 1 hour at 37° C.The procedure was stopped by the addition of 4.0 ml of GVBS⁻⁻centrifuged for 5 minutes at 1,620×g, and the absorbance of thesupernatants was read at 412 nm in a spectrophotometer. The percentlysis of each sample was standardized to that of the serum only control.

Results: Solubility of Sarcosine Substituted Peptides

TABLE 5 shows the solubility and hemolytic assay in factor B depletedserum of sarcosine (Sar) substitution peptides. The final concentrationof peptide in factor B-depleted serum was 0.77 mM. Each peptide wasevaluated in triplicate and the mean values are reported. Peptides notsoluble in water were re-suspended in DMSO. In the hemolytic assay,soluble peptides are standardized to water and insoluble peptidesstandardized to DMSO.

TABLE 5  Peptide Solu- Hemo- Name and Peptide Amino  SEQ  bility lysisControls Acid Sequence ID NO in water (%) Water — — 100.00 GVBS — — 1.31DMSO — — 95.13 PA IALILEPICCQERAA SEQ ID No 3.15 NO: 3 PA-I1Sar(Sar)ALILEPICCQERAA SEQ ID No 84.49 NO: 4 PA-A2Sar I(Sar)LILEPICCQERAASEQ ID Yes 37.72 NO: 5 PA-L3Sar IA(Sar)ILEPICCQERAA SEQ ID Yes 87.36NO: 6 PA-I4Sar IAL(Sar)LEPICCQERAA SEQ ID Yes 93.19 NO: 7 PA-L5SarIALI(Sar)EPICCQERAA SEQ ID Yes 85.13 NO: 8 PA-E6Sar IALIL(Sar)PICCQERAASEQ ID No 5.19 NO: 9 PA-P7Sar IALILE(Sar)ICCQERAA SEQ ID Yes 3.01 NO: 10PA-I8Sar IALILEP(Sar)CCQERAA SEQ ID Yes 33.92 NO: 11 PA-C9SarIALILEPI(Sar)CQERAA SEQ ID Yes 5.63 NO: 12 PA-C10Sar IALILEPIC(Sar)QERAASEQ ID No 56.18 NO: 13 PA-Q11Sar ALILEPICC(Sar)ERAA SEQ ID No 3.02NO: 14 PA-E12Sar IALILEPICCQ(Sar)RAA SEQ ID No 105.72 NO: 15 PA-R13SarIALILEPICCQE(Sar)AA SEQ ID No 3.42 NO: 16 PA-A14Sar IALILEPICCQER(Sar)ASEQ ID No 3.88 NO: 17 PA-A15Sar IALILEPICCQERA(Sar) SEQ ID No 21.68NO: 18

Peptide derivatives PA-P7Sar (SEQ ID NO: 10) and PA-C9Sar (SEQ ID NO:12) were both water soluble and inhibited complement activity to thesame degree as the PA peptide (SEQ ID NO: 3). A dose-dependentinhibition of complement activity by both PA-P7Sar and PA-C9Sar in ahemolytic assays is shown in FIG. 1.

Example 2—Solubility and Hemolytic Assay of PEGylated PA Peptides

Methods: Hemolytic Assay.

Polar Assortant peptides were serially diluted in undiluted factorB-depleted human sera (Complement Technologies, Inc.) and incubated for1 hour at 37° C. Water, GVBS⁺⁺ and DMSO were included as controls. Thesepeptides were then diluted with GVBS⁺⁺ to equal 2.5% serum, of which0.25 ml was combined with 0.4 ml of GVBS⁺⁺ and 0.1 ml of sensitizedsheep red blood cells (RBCs) and again incubated for 1 hour at 37° C.The procedure was stopped by the addition of 4.0 ml of GVBS⁻⁻,centrifuged for 5 minutes at 1,620×g, and the absorbance of thesupernatants was read at 412 nm in a spectrophotometer. The percentlysis of each sample was standardized to that of the serum only control.

Initially, PA was PEGylated with 24 PEG moieties on the N terminus(dPEG24-PA, SEQ ID NO: 20), on the C terminus (PA-dPEG24, SEQ ID NO:21), or on both the N terminus and C terminus (dPEG24-PA-dPEG24, SEQ IDNO: 19). All three PEGylated peptides were soluble in water and showedvarying degrees of complement inhibition with PA-dPEG24 (SEQ ID NO: 21)inhibiting complement activation to the same degree as PA (FIG. 2A).

As shown in FIG. 2B, PA-dPEG24 (SEQ ID NO: 21) inhibited complementactivation to the same degree as PA and displayed a broaderdose-response most likely due to its increased solubility in aqueoussolution. The structure of PA-dPEG24 (SEQ ID NO: 21) is shown in FIG. 3.

Next, PEGylated PIC1 derivatives were designed with decreasing numbersof PEG moieties on the C terminus. As shown in TABLE 6 below, most ofthese peptides retained solubility and complement inhibitory activity,with PA-dPEG24 (SEQ ID NO: 21) demonstrating the best inhibitoryactivity.

Results: Solubility of PEGylated Peptides

TABLE 6 shows the solubility and hemolytic assay in factor B depletedserum of PEGylated PA peptides. The final concentration of peptide infactor B-depleted serum was 0.77 mM. Peptides not soluble in water werere-suspended in DMSO. In the hemolytic assay, soluble peptides werestandardized to water and insoluble peptides standardized to DMSO.

TABLE 6  SEQ Solu- Hemo- Peptide  ID bility lysis name Peptide sequenceNO in water (%) Water — 100.00 DMSO — 95.17 PA-dPEG24IALILEPICCQERAA-dPEG24 21 Yes 2.99 PA-dPEG20 IALILEPICCQERAA-dPEG20 22Yes 11.41 PA-dPEG16 IALILEPICCQERAA-dPEG16 23 Yes 12.04 PA-dPEG12IALILEPICCQERAA-dPEG12 24 Yes 11.41 PA-dPEG08 IALILEPICCQERAA-dPEG08 25Yes 34.66 PA-dPEG06 IALILEPICCQERAA-dPEG06 26 No 44.82 PA-dPEG04IALILEPICCQERAA-dPEG04 27 Yes 12.36 PA-dPEG03 IALILEPICCQERAA-dPEG03 28Yes 12.57 PA-dPEG02 IALILEPICCQERAA-dPEG02 29 Yes 11.62

In vivo data in rats demonstrated that PA-dPEG24 (SEQ ID NO: 21)inhibits complement activation (as measured by red blood cell lysis in ahemolytic assay) by up to 90% within 30 seconds upon injection into ratswith inhibitory activity still observed up to 4 hours (FIG. 20). The twodoses were given intravenously (IV). This was compared to the vehiclecontrol (normal saline) which shows maximal hemolysis at all timepoints. PA-dPEG24 (SEQ ID NO: 21) was effective in vivo in 0.9% NaCl+10mM NaHPO₄, but also works in saline and likely other common aqueoussolutions (eg., Ringer's lactate, D5W, etc.) for IV infusion in humans.

Example 3—Solubility and Hemolytic Assay of PEGylated and Sarcosine(SAR) and/or Alanine Substitution Peptides

Table 7 shows the solubility and hemolytic assay in factor B depletedserum of PEGylated and sarcosine (SAR) substitution peptides. The finalconcentration of peptide in factor B-depleted serum was 0.77 mM.Peptides not soluble in water were re-suspended in DMSO. In thehemolytic assay, soluble peptides are standardized to water andinsoluble peptides standardized to DMSO.

TABLE 7  Solu- bi- SEQ lity Hemo- Peptide name ID in lysis and controlsPeptide sequence NO water (%) Water — — 100.00 DMSO — — 95.17 PA-dPEG24IALILEPICCQERAA-dPEG24 21 Yes 2.99 PA-C9SarC10A IALILEPI(Sar) A QERAA 30Yes 99.37 PA-C9SarD10 IALILEPI(Sar)QERAA 31 Yes 100.52 PA-P7SarC9SarIALILE(Sar)I(Sar)CQERAA 32 Yes 82.72 PA-E6Sar- IALIL(Sar)PICCQERAA- 33Yes 12.88 dPEG24 dPEG24 PA-Q11Sar- IALILEPICC(Sar)ERAA- 34 Yes 12.04dPEG24 dPEG24 PA-R13Sar- IALILEPICCQE(Sar)AA- 35 Yes 4.26 dPEG24 dPEG24PA-A14Sar- IALILEPICCQER(Sar)A- 36 Yes 3.68 dPEG24 dPEG24 E6SarP7SarIALIL(Sar)(Sar)ICCQERAA 37 Yes 3.57 E6SarC9Sar IALIL(Sar)PI(Sar)CQERAA38 Yes 6.21 Q11SarP7Sar IALILE(Sar)ICC(Sar)ERAA 39 Yes 3.22 Q11SarC9SarIALILEPI(Sar)C(Sar)ERAA 40 Yes 15.42 R13SarP7Sar IALILE(Sar)ICCQE(Sar)AA41 No 26.35 R13SarC9Sar IALILEPI(Sar)CQE(Sar)AA 42 No 84.81 A14SarP7SarIALILE(Sar)ICCQER(Sar)A 43 Yes 4.37 A14SarC9Sar IALILEPI(Sar)CQER(Sar)A44 No 14.73 E6AE12A- IALIL A PICCQ A RAA-dPEG24 45 Yes 4.95 dPEG24E6AE12AC9Sar IALIL A PI(Sar)CQ A RAA 46 No 2.99 E6AE12AP7Sar IALIL A(Sar)ICCQ A RAA 47 Yes 4.60

Changes were made to the PA peptide in which multiple amino acids weresubstituted with sarcosine and/or alanine. Some peptides were furthercombined with PEGylation. Multiple peptides retained the same level ofinhibitory activity and solubility as PA-dPEG24 (SEQ ID NO: 21),including, PA-R13Sar-dPEG24 (SEQ ID NO:35), PA-A14Sar-dPEG24 (SEQ ID NO:36), E6SarP7Sar (SEQ ID NO: 37), Q11SarP7Sar (SEQ ID NO: 39),A14SarP7Sar (SEQ ID NO: 43), E6AE12A-dPEG24 (SEQ ID NO: 45), andE6AE12AP7Sar (SEQ ID NO: 47).

Example 4—Minimum Inhibitory Concentration (MIC) for PIC1 Peptides

Methods: Minimum Inhibitory Concentration (MIC) Assay.

A microdilution minimum inhibitory concentration (MIC) assay wasperformed where increasing amounts of PA-dPEG24 (SEQ ID NO:21) orsarcosine-derivatives (SEQ ID NO: 4-18, 30-47) was added to variousbacteria growing in broth culture in a microtiter plate. The plates werethen incubated at 37° C. overnight and then the plates were read on aspectrophotometer at an absorbance of OD600 nm to determine theturbidity of the solution. Decreased turbidity is indicative ofinhibition of bacterial growth.

Results

PA-dPEG24 (SEQ ID NO: 21) was able to dose-dependently inhibitStaphylococcus aureus growth compared to a saline control (FIG. 4A)above 1 mM concentration and inhibited the same pathogen in a similarmanner to the antibiotic vancomycin (FIG. 4B). PA-dPEG24 (SEQ ID NO: 21)was able to inhibit Klebsiella pneumonia at >3 mg/ml similar togentamycin (FIG. 4C) and also inhibit Pseudomonas aeruginosa (FIG. 4D)at ≥13 mg/ml. The activity of PA-dPEG24 (SEQ ID NO: 21) was compared to3 water soluble sarcosine substitution peptides (PA L3Sar (SEQ ID NO:6), PA I4Sar (SEQ ID NO: 7) and PA L5Sar (SEQ ID NO: 8)) againstPseudomonas aeruginosa and it was found that all 3 sarcosine-derivativespeptides dose-dependently inhibited this species of bacteria at ≥6 mg/ml(FIG. 4E). These results demonstrate a completely novel antimicrobialproperty of these peptides which has not been previously reported thatis completely independent of the complement suppressing properties ofthe PIC1 family of peptides.

Next, it was tested whether PA-dPEG24 (SEQ ID NO: 21), PA L3Sar (SEQ IDNO: 6), PA I4Sar (SEQ ID NO: 7) and PA L5Sar (SEQ ID NO: 8) couldinhibit the growth of Gardnerella, a Gram variable, anaerobiccoccobacillus that is a common cause of bacterial vaginosis. All 4peptides inhibited the growth of Gardnerella (FIG. 5). It was alsoassayed whether PA-dPEG24 (SEQ ID NO: 21) could inhibitGardnerella-mediated complement activation. In this assay normal humanserum was incubated with Gardnerella in the presence of increasingamounts of PA-dPEG24 (SEQ ID NO: 21) for 1 hour at room temperature.Complement activation was assayed by measuring the production of C5ausing ELISA methodology. Increasing amounts of PA-dPEG24 (SEQ ID NO: 21)inhibited C5a formation (FIG. 6). These results suggest that PIC1peptides (e.g. SEQ ID NOs: 3-47) could be delivered locally in thevagina to treat bacterial vaginosis by inhibiting the replication ofGardnerella as well as Gardnerella-initiated inflammation. Theassociated inflammation causes the symptoms of bacterial vaginosis andincreases the risk of HIV-transmission by disrupting normal barrierdefenses in the vagina.

Example 5—Assay to Detect Complement-Mediated Hemolysis can DiscriminateErythrocyte Transfusions Likely to Cause Acute Transfusion Reactions(ATR)

Methods: Assay to Detect Complement-Mediated Hemolysis.

A 13 year old female B+ with sickle cell disease received three units ofO+ packed red blood cells (pRBCs) from two different donors. During thefirst transfusion of two units, the patient experienced hypotension (BP89/39) followed by fever (Tmax 102.6° F.). Transfusions of the thirdtransfused product was tolerated without complications. Due to suspicionfor an acute transfusion reaction (ATR), transfusion reaction work-up ofthe first transfused blood product revealed elevated anti-B titers.Subsequent investigative study of both identified donors indicated thefollowing: Donor #1 was a 48 year old male with no reported transfusionadverse events. Donor #2 was a 61 year old female with priorpregnancies, but no reported transfusion adverse events.

Plasma from both donors were diluted with GVBS++ (veronal bufferedsaline with 0.1% gelatin, 0.15 mM CaCl₂ and 1 mM MgCl₂) and incubatedwith purified erythrocytes from a B+ donor at 37° C. for 1 hour. Freehemoglobin was measured by spectrophotometer at 412 nm. Classicalcomplement activation was further evaluated by adding the classicalcomplement pathway inhibitor PA-dPEG24 (SEQ ID NO: 21) to the hemolyticplasma sample prior to incubation with erythrocytes.

Results

Complement mediated hemolysis was >100-fold different between the twoplasma samples from the different donors (TABLE 8). TABLE 8 shows plasmaanti-B titers and complement hemolysis for two O+ donors. Classicalcomplement activation was confirmed by blocking hemolysis with theclassical complement pathway inhibitor, PA-dPEG24 (SEQ ID NO: 21).

TABLE 8 Complement Hemolysis in Anti-B titer (plasma) hemolysis presenceof RT 37° C. IgG (Abs 412 nm) PA-dPEG24 Donor O+ #1 128 64 256 0.7470.008 Donor O+ #2 128 16 256 0.007

This data demonstrates that PIC1 peptides (e.g. SEQ ID NOs: 3-47) couldbe utilized to ascertain if ATRs are complement mediated. The data inTABLE 8 above is shown in FIG. 7 along with a dose-responsePA-dPEG24(SEQ ID NO: 21) inhibition study of the highly hemolytic plasmasample (FIG. 8). A dose-response inhibition of hemolysis wasdemonstrated with PA-dPEG24 (SEQ ID NO: 21) up to >95% inhibition. Thisshowed that the type-O RBC transfusion leading to ATR in the type-Brecipient was a classical complement-mediated event. FIG. 8 showed itwas possible to pharmacologically block the extremely robust hemolyticactivity mediated by this donor's plasma in this ATR in vitro model.

Conclusion

Past methodologies have not been able to adequately predict thelikelihood of complement-mediated ATR. In this case two units of type-ORBCs were given to a type-B recipient who then suffered ATR. There wasestimated to be about 30-40% plasma by volume in a unit of packed RBCs(pRBCs) (i.e. 150-200 ml plasma per 500 ml unit). The antibodies in theplasma of the RBC transfusions likely initiated classical pathwaycomplement-mediated ATR. The IgG titers for the plasma from donor ofeach RBC transfusion were both high, and thus non-predictive of thisplatelet transfusion caused the AHTR. A CH50-type complement hemolyticassay, however, was able to readily identify which type-O plasma causesmassive hemolysis of type-B erythrocytes (>100-fold increase).Therefore, a hemolytic complement assay performed before platelettransfusion would have identified that one of the units of platelets waslikely to cause AHTR and could have prevented this serious adverseevent.

Therefore, PIC1 peptides (SEQ ID NOS: 3-47) can be used to discriminaterisk of complement-mediated ATR before a blood transfusion. This dataalso supports the use of PIC1 peptides (SEQ ID NOS: 3-47) as a treatmentfor ATRs in various clinical scenarios.

Example 6—PIC1 (SEQ ID NO: 21) Enhances Survival of Human Erythrocytesin an Animal Model of Acute Intravascular Hemolytic Transfusion Reaction

Methods: Ethics Statement.

Adolescent male Wistar rats (200-250 g) with indwelling jugularcatheters were purchased from Harlan laboratories and used under theEastern Virginia Medical School (EVMS) IACUC (Institutional Animal Careand Use Committee) approved protocols 12-003 and 15-003. Central linemaintenance was followed as recommended by the animal vendor, withapproval from the EVMS IACUC.

After written informed consent was provided, a healthy human volunteerdonated AB blood which was used to generate purified red blood cells(RBCs) (EVMS IRB protocol 02-06-EX 0216). These RBCs were also used forin vitro analyses as the source of human RBCs (huRBC).

Methods: Human RBC Purification and Animal Experiments

Human RBCs (huRBCs) acquired the morning of the animal experiments wereprocessed as described in (Shah et al., “Complement inhibitionsignificantly decreases red blood cell lysis in a rat model of acuteintravascular hemolysis.” Transfusion. 2014). This process generates 1ml huRBCs at 80% hematocrit, which was given as a 15% transfusion to therats.

The AIHTR Wistar rat model was previously established in which cobravenom factor (CVF) was demonstrated to inhibit complement-mediated lysisof huRBCs, whereas animals receiving normal saline (NS) demonstratedclassical pathway, complement-mediated hemolysis. (Shah T A, Mauriello CT, Hair P S, et al. “Complement inhibition significantly decreases redblood cell lysis in a rat model of acute intravascular hemolysis.”Transfusion. 2014). Dosing of the PEGylated derivative, PA-dPEG24 (SEQID NO: 21) (New England Peptide, MA) was established in Wistar rats.FIG. 21A shows the sequence of events in a dosing experiment using a low(20 mg) and high dose (40 mg) of PA-dPEG24 (SEQ ID NO: 21) in the AIHTRanimal model. Animals were randomly assigned to various groups wherethey received either normal saline (n=6), 20 mg PA-dPEG24 (SEQ ID NO:21) (n=4) or 40 mg PA-dPEG24 (SEQ ID NO: 21) (n=7) before the huRBCtransfusion (prophylaxis arm). FIG. 21B shows the second set ofexperiments where one group of animals received 40 mg PA-dPEG24 (SEQ IDNO: 21) (n=8) before the huRBC transfusion (prophylaxis arm) and aseparate set of animals (n=10) receiving 40 mg PA-dPEG24 (SEQ ID NO: 21)after the huRBC transfusion (rescue arm). For both experiments, animalsallotted to the saline control group received NS prior to huRBCtransfusion whereas the CVF group received 130 μg CVF (ComplementTechnologies, Inc.) intra-peritoneum (I.P.) 24 hours prior to the huRBCtransfusion. In an additional experiment, separate groups of animalswere injected with 40 mg Immune globulin intravenous (POLYGAM®, BaxterHealthcare Corporation, CA) IVIG (n=3) and 40 mg PA-dPEG24 (SEQ ID NO:21) (n=3) before the huRBC transfusion (IVIG vs. PA-dPEG24).

Using weight-based doses of ketamine and acepromazine, animals weresedated throughout the course of the experiment with periodic monitoringof vital signs. Blood samples were collected into EDTA microtainer tubes(BD) from the animals prior to the huRBC transfusion and then at 30seconds, 5 minutes, 20 minutes, 60 minutes and 120 minutes after thehuRBC transfusion. In the group of animals receiving IVIG vs. PA-dPEG24(SEQ ID NO: 21), blood samples were drawn prior to the huRBC transfusionand then 30 seconds, 5 minutes, 20 minutes, 60 minutes, 120 minutes, 180minutes, 240 minutes, 300 minutes and 360 minutes after the huRBCtransfusion. These samples were kept shaking at room temperature priorto processing and centrifuged at 2655×g for five minutes to separate outthe plasma and sediment the RBCs. Plasma was aliquoted and the RBCpellet was processed separately as described below. Upon completion ofthe final (120 minutes or 360 minutes) blood draw, the animal waseuthanized using Fatal Plus (Vortech). A necropsy was completed tocollect organs (liver, spleen and bilateral kidney) for histopathology.In the experiment IVIG vs. PA-dPEG24 (SEQ ID NO: 21), bilateral kidneysacquired from each animal were weighted separately and stored informalin prior to processing and paraffin embedding. Hematoxylin andeosin (H&E) stained sections were reviewed by a blinded pathologist.

Methods: Flow Cytometry

Flow cytometry was performed on the collected RBCs using a FACS Caliburflow cytometer (Becton Dickinson, Franklin Lakes, N.J.) with DXP 8 Color488/637/407 Upgrade (Cytek Development, Fremont, Calif.). Samples werestained with either FITC and/or APC labeled antibodies. The data wasacquired using Cytek FlowJo CE version 7.5.110.6. Approximately 1×10⁵and 5×10⁵ events per sample were gathered for single and double labeledflow, respectively. Data was analyzed using FlowJo X version 10.0.7r2(FlowJo LLC, Ashland, Oreg.). Although FITC and APC are not expected tocause much spillover, digital compensation was used in the analysis.

Single labeled flow: The RBC pellet collected after separating theplasma was washed, diluted and stained with FITC-conjugated anti-humanCD235a (glycophorin A, eBioscience, San Diego, Calif.) at 1:200 inGVBS−− (veronal-buffered saline (VBS) with 0.1% gelatin, 0.01 mol/LEDTA) over 20 min while shaking at room temperature to minimizeagglutination. An antibody control consisted of the RBC pellet washed,diluted and unstained or stained with mouse IgG2b Iso Control FITC at1:200 (eBioscience, San Diego, Calif.).

Dual labeled flow: The RBC pellet generated above was washed and doublestained with APC-conjugated mouse anti-human CD235a (glycophorin A, BDBioscience, San Jose, Calif.) at 1:50 and FITC-conjugated goat-anti-ratC3 (MP Biomedical, Santa Ana, Calif.) at 1:200 in GVBS−− for 20 min atroom temperature. Three controls consisting of unstained RBCs, RBCsacquired at 30 sec stained with the APC tagged antibody and FITC taggedantibody were used for quadrant analysis.

In vitro flow cytometry experiment: 100% complement sufficient Wistarrat sera (Innovative Research, Novi, Mich.) was incubated with PA-dPEG24(SEQ ID NO: 21) at 12 mg/ml (4 mMol) for 5 minutes at room temperatureto generate PA-dPEG24 (SEQ ID NO: 21) treated serum. HuRBC's prepared asabove were then incubated with PA-dPEG24 (SEQ ID NO: 21) treated serumor with 100% complement sufficient Wistar rat sera untreated serum for 5min each at 37° C. The cells were then washed twice with GVBS toterminate complement activation. These cells were then double stainedwith APC-conjugated mouse anti-CD235a and FITC-conjugated goat anti-C3as described above. After washing, the cells were combined with 85%non-opsonized, non-stained cells to mimic the 15% transfusion used invivo and analyzed by flow cytometry using the same conditions stated indual labeled flow, above.

Methods: Hemoglobin and Bilirubin Measurements

Plasma generated from the above experiments was analyzed for the freehemoglobin using spectrophotometry, as described previously (Shah et al.“Clinical hypothermia temperatures increase complement activation andcell destruction via the classical pathway.” J Transl Med 2014;12:181.). For bilirubin measurements, the pre-bleed and 120 minuteplasma samples from the prophylaxis (n=8) and NS (n=7) groups wereanalyzed for the amount of bilirubin present using the Bilirubin AssayKit (Sigma-Aldrich, St. Louis, Mo.) in half the manufacturer's recommendvolume. Due to large amounts of hemolysis in the latter time points andthe associated optical interference in bilirubin analysis, all thesamples were pre-treated with HemogloBind™ (Biotech, NJ) prior toanalysis with the Bilirubin Assay Kit. (Koseoglu M, Hur A, Atay A,Cuhadar S. “Effects of hemolysis interferences on routine biochemistryparameters.” Biochemia medica. 2011; 21(1): 79-85].

Results

PA-dPEG24 (SEQ ID NO: 21) blocked AIHTR in rats when givenprophylactically before the transfusion, as well as when PA-dPEG24 (SEQID NO: 21) was administered as rescue treatment after transfusion.PA-dPEG24 (SEQ ID NO: 21) was compared in a dose comparable fashionagainst intravenous immunoglobulin (IVIG) in the AIHTR disease animalmodel. IVIG is being used in clinical practice in conjunction withphototherapy to reduce the need for exchange transfusion in neonateswith hyperbilirubinemia due to ABO incompatibility related hemolyticdisease [Schwartz H P, Haberman B E, Ruddy R M. “Hyperbilirubinemia:current guidelines and emerging therapies.” Pediatric emergency care.2011; 27(9):884-889; “Management of hyperbilirubinemia in the newborninfant 35 or more weeks of gestation.” Pediatrics. 2004; 114(1):297-316;Cortey A, Elzaabi M, Waegemans T, Roch B, Aujard Y. “Efficacy and safetyof intravenous immunoglobulins in the management of neonatalhyperbilirubinemia due to ABO incompatibility: a meta-analysis.”Archives de pediatric: organe officiel de la Societe francaise depediatric. 2014; 21(9): 976-983].

Example 7—PIC1 (SEQ ID NO: 21) Enhances Survival of Human Erythrocytesin an Animal Model of Acute Intravascular Hemolytic TransfusionReaction—Prophylactic Administration of SEQ ID NO: 21 Reduces Hemolysisof huRBCs in the AIHTR Model

Previous work had established the AIHTR model in Wistar rats anddemonstrated that a 15% transfusion of HuRBC resulted in classicalpathway-mediated intravascular lysis of the xenotransfused cells [Shahet al. “Complement inhibition significantly decreases red blood celllysis in a rat model of acute intravascular hemolysis.” Transfusion.2014 November; 54(11):2892-900.] “Clinical hypothermia temperaturesincrease complement activation and cell destruction via the classicalpathway.” J Transl Med 2014; 12:181.]. Depletion of complement activityby CVF led to increased survival of HuRBC as shown by flow cytometry andfree hemoglobin measurements with the transfused cells eventually beingsequestered in the spleen and liver [Shah et al. “Complement inhibitionsignificantly decreases red blood cell lysis in a rat model of acuteintravascular hemolysis.” Transfusion. 2014 November; 54(11):2892-900.].To assess the efficacy of PA-dPEG24 (SEQ ID NO: 21) inhibition on thesurvival of huRBCs in this AIHTR model, animals were prophylacticallytreated with PA-dPEG24 (SEQ ID NO: 21) at doses of 20 and 40 mg/animalfollowed by 15% xenotransfusion of HuRBCs and blood draws at theindicated time points (FIG. 22A). Animals pre-treated with CVF served asa positive control for complement depletion and a separate set ofanimals received NS (vehicle control). Plasma isolated from NS-treatedanimals demonstrated a spike of hemolysis as assayed by released freehemoglobin (FIG. 22A). In contrast, animals pre-treated with CVF had lowlevels of free hemoglobin as previously reported (FIG. 22A) [Shah et al.“Complement inhibition significantly decreases red blood cell lysis in arat model of acute intravascular hemolysis.” Transfusion. 2014 November;54(11):2892-9004 Animals receiving both doses of PA-dPEG24 (SEQ ID NO:21) showed decreased levels of free hemoglobin similar to that observedfor CVF-treated animals; animals receiving 40 mg PA-dPEG24 (SEQ ID NO:21) showed significant reduction of free hemoglobin compared to NScontrol animals at 5 minutes and 20 minutes (P≤0.05) (FIG. 22A). Toverify the complement-mediated destruction of the transfused huRBC, RBCsfrom blood collected at the indicated time points were isolated andlabeled with antibody to human glycophorin A (CD235a) and analyzed byflow cytometry. Animals receiving 20 or 40 mg doses of PA-dPEG24 (SEQ IDNO: 21) showed survival of transfused cells comparable to that observedin the CVF treated animals up to 20 minutes (FIG. 22B). There was nostatistically significant difference observed between the effects causedby both doses of PA-dPEG24 (SEQ ID NO: 21) and CVF in these experiments.These results show the protective effect of PA-dPEG24 (SEQ ID NO: 21) onthe survival of incompatible HuRBCs in the animal model of AIHTR.

Example 8—PIC1 (SEQ ID NO: 21) Enhanced Survival of Human Erythrocytesin an Animal Model of Acute Intravascular Hemolytic TransfusionReaction—Administration of SEQ ID NO: 21 after HuRBC TransfusionProtects the Transfused Cells from Complement-Mediated Hemolysis

PA-dPEG24 (SEQ ID NO: 21) was able to show protection of the HuRBC whenadministered to rats prior to xenotransfusion, It was also discoveredthat PA-dPEG24 (SEQ ID NO: 21) given immediately after xenotransfusioncould protect the HuRBCs from complement-mediated attack. Rats receiveda 40 mg dose of PA-dPEG24 (SEQ ID NO: 21) either 30 seconds before(prophylaxis arm) or 30 seconds after xenotransfusion of HuRBCs(treatment/rescue arm). A separate group of animals also received NSvehicle control or were pre-treated with CVF (FIG. 21B). The protectiveeffect of 40 mg PA-dPEG24 (SEQ ID NO: 21) given before the transfusion(prophylaxis group) and after the transfusion (treatment/rescue group)on the amount of hemolysis as assayed by free hemoglobin wassignificantly reduced compared to the animals who received NS at 5minutes (P≤0.005) and 20 minutes (P≤0.005) (FIG. 23A), representing thegreatest amount of hemolysis in this AIHTR model (FIGS. 22A & 23A) FIG.23B shows the cumulative hemolysis occurring in the various groups ofanimals (Saline, Prophylaxis, Treatment and CVF). Both the treatment andprophylaxis groups demonstrated reduced hemolysis compared to the salinegroup of animals (P<=0.05). Another common pathway of hemoglobinmetabolism when it is released into the plasma as a result ofintravascular hemolysis is indirect bilirubinemia [Strobel E. “HemolyticTransfusion Reactions.” Transfusion medicine and hemotherapy:offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin andImmunhamatologie. 2008; 35(5):346-353]. NS treated animals showed alarge increase in free bilirubin between the pre-bleed and 120 minutetime point (FIG. 23C), consistent with RBC lysis as assessed by freehemoglobin and flow cytometry. In contrast to the NS treated animals, 40mg PA-dPEG24 (SEQ ID NO: 21) given prior to the transfusion of HuRBCssignificantly reduced the amount of bilirubin circulating at 120 minutescompared to the animals receiving NS (P=0.0015) (FIG. 23C).

Example 9—Peptide Inhibitor of Complement C (SEQ ID NO: 21) EnhancesSurvival of Human Erythrocytes in an Animal Model of Acute IntravascularHemolytic Transfusion Reaction Assessment of Complement Deposition onTransfused HuRBCs

The results described above demonstrate that PIC1 (SEQ ID NO: 21) can beutilized in both a prophylactic and treatment strategy to preventhemolysis of transfused huRBC as assayed by huRBC survival, freehemoglobin levels and bilirubin release. To gain a more completeunderstanding of the role of complement deposition on the transfusedcells, an analysis of C3 binding to huRBCs was conducted. To establishexperimental conditions, an in vitro study was performed using HuRBCsexposed to rat serum followed by analysis of C3 deposition on the cellsby flow cytometry. Rat serum treated with and without PA-dPEG24 (SEQ IDNO: 21) was added to HuRBCs and allowed to opsonize for 5 minutes. Cellswere then analyzed by dual labeled flow cytometry with representativeplots for HuRBCs exposed to untreated or PA-dPEG24 (SEQ ID NO:21)-treated serum shown in FIGS. 24A and 24B, respectively.

Quantification of HuRBCs opsonized in rat serum with and withoutPA-dPEG24 (SEQ ID NO: 21) showed a relative increase in the number ofcells with no C3 deposition in PA-dPEG24 (SEQ ID NO: 21)-treated serum(FIG. 24C). When the fraction of dual labeled cells relative to thetotal number of glycophorin A labeled cells was calculated, there wasa >1.5 fold reduction in C3 deposition on cells incubated with PA-dPEG24(SEQ ID NO: 21)-treated serum versus untreated serum (FIG. 24D). Inuntreated serum, a larger population of dual labeled cells (Q2) wasobserved to shift over to Quadrant 1 which represented cells with C3deposition. These findings demonstrated that PA-dPEG24 (SEQ ID NO: 21)rescued HuRBCs from opsonization and impending hemolysis throughactivation of the complement proteins in vitro.

To evaluate the effect of PA-dPEG24 (SEQ ID NO: 21) on C3 deposition oftransfused HuRBCs in the AIHTR model, RBCs isolated from the blood ofanimals that received either CVF, NS or PA-dPEG24 (SEQ ID NO: 21) priorto xenotransfusion or post-xenotransfusion were subject to two colorflow cytometry for both human glycophorin A expression and rat C3.Representative flow cytometry plots of RBCs at 30 seconds, 5 minutes and20 minutes post-transfusion are shown for NS (FIGS. 25A-C) and PA-dPEG24(SEQ ID NO: 21) treated (FIG. 25D-F) animals. NS-treated animalsexhibited an increased number of double-positive cells over timecompared to PA-dPEG24 (SEQ ID NO: 21)-treated animals. Conversely,PA-dPEG24 (SEQ ID NO: 21)-treated animals had more glycophorin Apositive cells consistent with increased RBC survival as assessed bysingle label flow cytometry and free hemoglobin measurements (FIGS.22A-B & 23A-C). To more precisely quantify these results, the numbers ofevents captured by flow cytometry were analyzed and graphed (FIG. 26A).At 30 seconds after HuRBC transfusion prophylactic treatment withPA-dPEG24 (SEQ ID NO: 21) showed a dramatic increase in the numbers ofcirculating HuRBCs (Human—No C3) compared with no intervention (NS) oreven CVF-treated animals (FIG. 26A). The amount of surviving HuRBCs at30 seconds for the PA-dPEG24 (SEQ ID NO: 21) treatment group was similarto the animals receiving NS, which was expected as PA-dPEG24 (SEQ ID NO:21) was given 30 seconds after the transfusion (FIG. 26A). At 5 and 20minutes after transfusion, groups of animals receiving prophylaxis andtreatment with PA-dPEG24 (SEQ ID NO: 21) showed decreased C3 depositionon the surface of circulating HuRBCs compared with animals receiving nointervention (NS) (FIG. 26B). For animals receiving PA-dPEG24 (SEQ IDNO: 21) after transfusion (treatment arm), while many HuRBCs werecleared from the circulation prior to PA-dPEG24 (SEQ ID NO: 21)treatment (FIG. 26A), HuRBCs persisted in circulation through 20 minutesat higher numbers than that observed for the NS group (FIG. 26C). Inorder to evaluate complement attack of innocent bystander RBCs (i.e. ratRBCs), C3-fragment bound RBCs that were not labeled withanti-glycophorin A were counted. Prophylactic administration ofPA-dPEG24 (SEQ ID NO: 21) decreased innocent bystander attack ofnon-glycophorin A RBCs at all observed time points compared to the NScontrol animals. Post-transfusion treatment with PA-dPEG24 (SEQ ID NO:21) decreased ‘innocent bystander’ attack at the 5 and 20 minute timepoints (FIG. 26D).

Example 10—Investigation of Complement Effectors of Inflammation inCystic Fibrosis Lung Fluid—Complement Anaphylatoxins in CF Lung Fluid

Methods: Ethics Statement.

Sputum samples were obtained from consented patients as part of theirstandard of care visit at the Children's Hospital of The King'sDaughters Cystic Fibrosis Center and collected from the ClinicalMicrobiology Laboratory prior to being discarded. This was performedunder an Eastern Virginia Medical School IRB approved protocol12-08-EX-0200. Samples were given a numerical code that was linked in anencrypted file to the clinical database. Control sputum samples wereobtained from healthy human volunteers.

Methods: Sputum Sols

Sputum samples were obtained as expectorated sputum and placedimmediately on ice. To confirm that induction of sputum did not altercomplement in the sol, a control experiment was performed with a healthyhuman volunteer who produced an expectorated sputum and an inducedsputum showing no differences in complement activation or complementeffector concentrations. The soluble (sol) fraction was generated bycold (4° C.) centrifugation at 14,000 g for 60 minutes and recovery ofthe free flowing liquid fraction, similar to methods previouslydescribed [Davies J R, Svitacheva N, Lannefors L, et al. “Identificationof MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosisairway secretions.” Biochem J 1999; 344 Pt 2:321-30]. The sols werealiquoted and frozen at −80° C.

Clinical Data

Clinical Data were obtained from data entered into Port C F for theclinic visit at which the sputum sample was collected and from review ofthe medical record. The FEV1% predicted value was obtained frompulmonary function testing performed at the clinic visit when the sputumsample was collected. The bronchiectasis score was based on the mostrecent radiographic lung study prior to obtaining the sputum sample,usually plain radiograph. A bronchiectasis score was assigned asfollows: 0=normal; 1=1 lobe, mild; 2=2-4 lobes; 3=all lobes. Cysticfibrosis related diabetes (CFRD) status was based on the most recentendocrinology assessment prior to obtaining the sputum sample. CFRDstatus was scored as follows: 0=normal; 1=glucose intolerance; 2=CFRD.Part of each sputum sample was sent for microbiologic testing in theclinical microbiology laboratory at CHKD and organisms were recorded.The organisms were categorized as to whether the following were presentor absent: Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderiacepacia complex, or Candida species. Patient medications at the time ofclinic visit were also recorded. Medications were categorized as towhether the following were present or absent: systemic corticosteroid,inhaled corticosteroid, azithomycin, inhaled antibiotic, or systemicantibiotic (excluding azithromycin).

Methods: ELISAs & Western Blots

The C5a concentration in sputum sol was quantitated using a C5a ELISAkit (R&D Systems). C3a and C4a from sputum sol were both measured usingtheir respective ELISA kits (BD Biosciences). Bound C3-fragments weredetermined using a total C3 ELISA. Briefly, a goat anti-human C3antibody (Complement Technology) was used to coat flat-bottom Immulon-2plates the night before. The plates were then washed with PBST (PBS with0.1% TWEEN), blocked for two hours with 3% BSA/PBS, washed again, andfollowed by the addition of samples and a pure C3 standard (ComplementTechnology) for 1 hour in block buffer. Plates were washed, incubatedwith a chicken anti-human C3 antibody (Sigma) for 1 hour, washed again,and incubated with a goat anti-chicken HRP antibody (Genway). Plateswere developed with TMB Substrate Solution (Thermo Scientific) andstopped with 2.5 N H2504 (Hair P S, Echague C G, Rohn R D, et al.“Hyperglycemic conditions inhibit C3-mediated immunologic control ofStaphylococcus aureus.” J Transl Med 2012; 10:35]. Bound C4-fragmentswere evaluated with the same ELISA method as for C3, except using a goatanti-human C4 antibody to coat the plates, a pure C4 as the standard(both Complement Technologies), and a chicken anti-human C4 antibody(Abcam) as the primary antibody.

C5a fragments were analyzed by Western Blot using a mouse anti-human C5aantibody (R&D Systems) to probe followed by a goat anti-mouse HRPantibody (Sigma) and detected with ECL.

Results

To evaluate whether complement anaphylatoxins were elevated in CF lungfluid, CF sols from the sputum of CF patients were assayed for eachcomplement anaphylatoxin and compared with sputum sols from healthyhuman controls. The most inflammatory complement anaphylatoxin was C5a,which was assayed by ELISA and Western blot. Mean C5a concentration inCF sols was 4.8-fold higher (P=0.04) compared with the mean for healthycontrols (FIG. 10A). Qualitative analysis by Western blot probing forC5a confirmed that large amounts of C5a were present in CF sols comparedwith controls (FIG. 10B). C3a is a complement effector that is generatedduring activation of the central complement component C3. Mean C3aconcentrations in CF sols was 4-fold higher (P=0.03) compared withcontrols (FIG. 10C). C4a is the least potent of complementanaphylatoxins and is generated during classical or lectin pathwaycomplement activation. Mean C4a concentration was 2-fold higher in CFsols (P=0.05) compared with controls (FIG. 10D). Together these datashow that the concentration of complement anaphylatoxins in CF lungfluid was significantly elevated, suggesting significant complementactivation in CF lung fluid. The potent ability of C5a to recruitneutrophils and stimulate degranulation [Lambris J D, Sahu A, Wetsel RA. “The chemistry and biology of C3, C4, and C5. In: Volanakis J E,Frank M M, (eds).” The human complement system in health and disease.New York: Marcel Dekker; 1998, 83-118; Mollnes T E, Brekke O L, Fung M,et al. “Essential role of the C5a receptor in E coli-induced oxidativeburst and phagocytosis revealed by a novel lepirudin-based human wholeblood model of inflammation.” Blood 2002; 100(5):1869-77; Laursen N S,Magnani F, Gottfredsen R H, et al. “Structure, function and control ofcomplement C5 and its proteolytic fragments.” Curr Mol Med 2012;12(8):1083-971 suggested that C5a could contribute to the highconcentrations of neutrophil elastase in CF lung fluid, which isassociated with parenchymal destruction. Additionally, the elevatedlevels of C4a in CF lung fluid, suggested that much of the complementactivation occurring in CF lung fluid may be occurring via the classicalor lectin complement pathways.

Example 11—Investigation of Complement Effectors of Inflammation inCystic Fibrosis Lung Fluid—Complement Opsonization of Staphylococcusaureus in CF Lung Fluid

Methods: S.Aureus Opsonization with CF Sols

Staphlycoccus aureus strain Reynolds was grown in 2% NaCl Columbia brothat 37° C. to log phase, washed twice with GVBS' (vernal buffered salinewith 0.1% gelatin, 0.15 mM CaCl₂, and 1.0 mM MgCl₂), and resuspended to1×10⁹ cells/ml. An equal volume of bacteria and CF or control sol wereincubated for one hour at 37° C. The bacteria were washed twice withGVBS⁻⁻ (VBS with 0.1% gelatin and 0.01 M EDTA), and then stripped ofbound complement fragments using methylamine, as previously described[Cunnion K M, Lee J C, Frank M M. “Capsule production and growth phaseinfluence binding of complement to Staphylococcus aureus.” Infect Immun2001; 69(11): 6796-803]. Results

In order to evaluate whether complement in CF lung fluid couldadequately opsonize a pathogenic bacteria, CF sol opsonization ofStaphylococcus aureus was evaluated. Given the large amounts ofcomplement activation that had already occurred in the CF lung fluids,it was important to determine whether there was residualcomplement-mediated host defense. Staphylococcus aureus were incubatedwith CF or control sols, washed and stripped of bound C3-fragments andbound C4-fragments. Staphylococcus aureus was robustly opsonized in CFsol yielding a nearly identical mean level of bound C3-fragmentscompared with normal controls (FIG. 11A). Staphylococcus aureus was alsorobustly bound by C4-fragments (P=0.13) with a non-significant trendtowards increased mean C4-fragment binding by CF sols compared withnormal controls (FIG. 11B). These results suggested that CF lung fluidretained a normal capacity to opsonize bacteria, suggesting that thisfacet of host defenses is not compromised. These results also show thatdespite significant complement activation having occurred, as evident byvery high anaphylatoxin levels, significant complement remains in CFlung fluid. This suggested a cycle of complement activation andrepletion consistent with persistent inflammation. The robustopsonization with C4-fragments suggested that the classical or lectincomplement pathway was active in CF lung fluid and may be thepredominant pathway of complement activation.

Example 12—Investigation of Complement Effectors of Inflammation inCystic Fibrosis Lung Fluid—C5a Generation in CF Lung Fluid byPseudomonas aeruginosa and Staphylococcus aureus

Pseudomonas aeruginosa was acquired as a discarded de-identifiedclinical isolate. It was grown in Mueller Hinton broth to log phase andwashed and re-suspended as was done with the Staphylococcus aureus. Bothbacteria were gently heat killed by incubating in a 70° C. water bathfor 15 minutes. Samples were plated to confirm that the bacteria weredead. CF or control sols were incubated with either live or deadPseudomonas aeruginosa or Staphylococcus aureus at equal volumes for onehour at 37° C. Afterward, the samples were spun at 14,000×g for 5minutes and the supernatant was collected and analyzed for C5a. Equalvolume of CF sol and PA-dPEG24 (SEQ ID NO: 21) [Shah et al. “Clinicalhypothermia temperatures increase complement activation and celldestruction via the classical pathway.” J Transl Med 2014; 12:181;Mauriello C T, Pallera H K, Sharp J A, et al. “A novel peptide inhibitorof classical and lectin complement activation including ABOincompatibility.” Mol Immunol 2012; 53(1-2):132-9], at 50 mg/ml, orsaline for controls, were combined and pre-incubated for 30 minutes atroom temperature. Afterward, 5×10⁷ CFU heat-killed Pseudomonasaeruginosa were added and incubated at 37° C. for 30 minutes. Thesamples were then spun at 14,000×g for 2 minutes and the supernatant wascollected and assayed in the C5a ELISA.

In order to evaluate whether CF lung fluid challenged with pathogenicbacteria commonly present in CF lungs would generate new C5a, the mostinflammatory anaphylatoxin, CF sols were incubated with live and deadPseudomonas aeruginosa and Staphylococcus aureus. Live and dead versionsof each bacteria were tested because both forms are likely to be presentin an infected CF lung. Additionally, secreted factors or adaptivechanges that could be produced by live bacteria would also alter C5ageneration. Before and after incubation with bacteria, C5aconcentrations were measured in CF to determine if new C5a weregenerated. Incubation of CF sols with live or dead Pseudomonasaeruginosa lead to an average increase in C5a generation of 2.3-fold(P=0.02). Similarly, incubation with live or dead Staphylococcus aureuslead to an average increase in C5a generation of 2.4-fold (P=0.02) (FIG.12A). These data also showed a difference in C5a generation between liveor dead Pseudomonas aeruginosa (P=0.05), but not between live or deadStaphylococcus aureus. This relationship between live or deadPseudomonas aeruginosa challenge appeared consistent for the differentCF sol samples (FIG. 12B), but there was no consistent relationshipbetween live or dead Staphylococcus aureus challenge (FIG. 12C). Thesedata showed that Pseudomonas aeruginosa and Staphylococcus aureus bothprovoke robust generation of the highly inflammatory C5a anaphylatoxinin CF lung fluid, suggesting that the presence of these bacteria in theCF lung may be enhancing inflammation and subsequent host tissue damage.Pseudomonas aeruginosa may have some ability to moderate C5a generationin CF lung fluid compared to dead Pseudomonas aeruginosa.

In order to evaluate the likely complement pathways by which Pseudomonasaeruginosa was activating C5a generation, an inhibitor of classical andlectin complement pathway activation was tested. Peptide inhibitors ofcomplement C1 are small peptide inhibitors of the classical and lectinpathways of complement activation CF sols were incubated in bufferalone, with dead Pseudomonas aeruginosa, or with PA-dPEG24 (SEQ ID NO:21) and dead Pseudomonas aeruginosa (FIG. 12D). Dead Pseudomonasaeruginosa increased C5a concentration by 1.6-fold compared toincubation of the CF sol in buffer alone. Addition of PA-dPEG24 (SEQ IDNO: 21) to the CF sol decreased C5a generation by Pseudomonas aeruginosa(P=0.001) to a level not significantly different from CF sol alone(P=0.22). Thus, addition of a classical/lectin pathway inhibitor blockedC5a generation by Pseudomonas aeruginosa, suggesting that Pseudomonasaeruginosa activates complement and C5a generation via the classical orlectin complement pathways. This finding was congruent with elevated C4alevels and robust C4-fragment opsonization.

Example 13—Investigation of Complement Effectors of Inflammation inCystic Fibrosis Lung Fluid—Complement Anaphylatoxin Correlation withClinical Characteristics

Methods: Statistical Analysis of Clinical Measures with C5a/C3a and ofLaboratory Data

The data were analyzed using SAS 9.4 (SAS Institute, Cary, N.C.) andSPSS 19 (SPSS Inc., Chicago, Ill.) software. The level of significancewas set at 0.05. Pearson and Spearman correlation coefficients werereported, where appropriate, for C5a level, C3a level, and the clinicalmeasures. Descriptive statistics were reported for C5a and C3a levelstratified by clinical measures. Simple linear regression andMann-Whitney U tests were used, where appropriate, to determineassociations between C5a level, C3a level, and the clinical measures. Amultivariable linear regression model for FEV1% was used to determineassociations with C5a level and C3a level.

Medians, quartiles, and 90^(th) percentiles were calculated using PSIplot. Means and standard error of the means (SEM) were calculated fromindependent experiments. Statistical comparisons were made usingStudent's t-test where appropriate with a level of significance set at0.05.

Results

CF sputum samples were collected from 15 patients. The clinicalcharacteristics of these 15 subjects is shown in TABLE 9. The subjectsspan a wide range of ages from 2-65 years old with a median age of 19.Median FEV1% predicted was 59; for children (n=7) the median BMI was 48%and for adults (n=4) the median BMI was 23.87 mg/kg². Clinical datacollected at the time of sputum sampling was obtained for a wide rangeof measures including FEV1% predicted, BMI percentage (children),bronchiectasis, CFRD status, pathogenic microorganisms cultured from thesputum and medications (i.e. corticosteroids, azithromycin, andantibiotics). Statistical correlations were assessed between theanaphylatoxins, C5a and C3a, and the clinical measures, see TABLE 10.Increased concentration of the highly inflammatory C5a positivelycorrelated with increased age (r=0.53, P=0.04), as shown in FIG. 13A.Increased C5a concentration correlated inversely with BMI percentile inchildren (r=−0.77, P=0.04) as shown in FIG. 13B. Increased C3a levelspositively correlated with increased FEV1% predicted (rs=0.63, P=0.02),as shown in FIG. 13C. Microorganisms (i.e. Pseudomonas aeruginosa,Burkholderia cepacia, Staphylococcus aureus, or yeast) recovered fromthe sputum, corticosteroids, azithromycin, or antibiotics did notcorrelate with levels of C5a or C3a. These results showed thatincreasing C5a concentration correlated with decreased BMI percentile inchildren, suggesting that increased complement inflammatory C5a in lungfluid may be associated with poorer overall health in children with CF.C3a level positively correlated with FEV1% predicted, suggesting apotentially protective effect from C3a on CF lung function.

TABLE 9 Characteristics of the CF subjects. Median (range) Age, y 19(2-65) Gender, % female 60 Child BMI, % 26 (10-70) Adult BMI 23.8(21.8-25.9) FEV1 % 59 (23-99)

TABLE 10 Correlation coefficients for complement effectors and clinicalmeasures C5a (ng/ml) C3a (ng/ml) Correlation coefficient Age, y 0.53^(a)−0.12^(b) P value (H₀: rho = 0) 0.04 0.68 N 15 14 Correlationcoefficient Child BMI, % −0.77^(a) −0.39^(b) P value (H₀: rho = 0) 0.040.38 N 7 7 Correlation coefficient FEV1 % 0.04^(a) 0.63^(b) P value (H₀:rho = 0) 0.89 0.02 N 14 13 Correlation coefficient Bronchiectasis0.12^(b) 0.15^(b) P value (H₀: rho = 0) Score 0.68 0.61 N 15 14Correlation coefficient CFRD Score 0.27^(b) 0.01^(b) P value (H₀: rho =0) 0.38 0.98 N 13 12 Bronchiectasis score: 0 = normal; 1 = 1 lobe, mild;2 = 2-4 lobes; 3 = all lobes CFRD score: 0 = normal; 1 = glucoseintolerance; 2 = CFRD ^(a)Pearson correlation coefficient ^(b)Spearmancorrelation coefficientExample 14—Modulation of C1q Interaction with C1q Receptors—Binding toCalreticulin ReceptorMethods

To demonstrate that PA blocks binding to the calreticulin receptor, thefollowing experiment was performed. Recombinant calreticulin or BSA(negative control) was used to coat a microtiter plate. C1q was thenadded to the plate alone was incubated with increasing amounts of PApeptide (SEQ ID NO: 3) and then assessed for binding to calreticulin. Asa negative control, a 15 amino acid peptide (CP2) that does not bind C1qwas used.

The interaction of labeled C1q with Raji cells was tested by confocalmicroscopy. Labeled C1q was added to Raji cells. 1.4125×10⁶ cells/mlwere fixed with 4% paraformaldehyde for 5 min and stored over-night at4° C. 1×10⁵ cells stained with: 1:40 C1q 488 for 20 min, washed ×2, andstained with DAPI mounting medium, and stored over-night at 4° C.

PA-dPEG24 (SEQ ID NO: 21) was pre-incubated with C1q at two differentconcentrations before adding to Raji cells. 1.4125×10⁶ cells/ml werefixed with 4% paraformaldehyde for 5 minutes and stored over-night at 4°C. 1×10⁵ cells stained with: 1:40 C1q 488 plus 1.45 mg/ml PA-dPEG24 (SEQID NO: 21) (1:20 of 29 mg/ml) or 2.9 mg/ml PA-dPEG24 (SEQ ID NO: 21)(1:10 of 29 mg/ml) for 20 minutes, washed twice, stained with DAPImounting medium, and stored overnight 4° C.

Results

PA-dPEG24 (SEQ ID NO: 21) blocked C1q binding to a specific C1q cellreceptor (calreticulin/cC1qR) in an in vitro assay and also blockedbinding of C1q to Raji cells (an immortalized B lymphocyte cell linethat is used for evaluating C1q interactions with C1q receptors).

C1q was added to a plate coated with calreticulin, the results showedthat C1q binds specifically to calreticulin but not BSA, as expected(FIG. 15). When plates were coated with C1q, calreticulin and BSA, PApeptide (SEQ ID NO: 3) bound specifically to C1q, the interactionbetween PA was specific for C1q and not calreticulin or BSA (FIG. 14A).When C1q was incubated with increasing amounts of PA peptide (SEQ ID NO:3) and then assessed for binding to calreticulin, PA dose-dependentlyinhibited binding to calreticulin (FIG. 16). As a negative control, a 15amino acid peptide (CP2) that does not bind C1q did not show competitiveinhibition of C1q binding to calreticulin (FIG. 16). Additionally, PA(SEQ ID NO: 3) inhibited C1q binding to purified calreticulin (FIG. 14B)and Raji cells (FIG. 14C-D) consistent with inhibition of C1q binding tosurface CRT. The experiment setup is show in FIG. 14E.

The interaction of labeled C1q with Raji cells was investigated byconfocal microscopy. Labeled C1q (FITC) bound to Raji cells (DAPI) asshown in FIGS. 17A-C. When PA-dPEG24 (SEQ ID NO: 21) was preincubatedwith C1q at two different concentrations, the amount of labeled C1qbound to Raji cells decreased significantly (FIGS. 18A-C and 19A-C),demonstrating that (SEQ ID NO: 21) inhibited binding of C1q to thesecells. Although not bound by any theory, (SEQ ID NO: 21) presumablyinhibited binding of C1q by disrupting C1q interaction with a C1qreceptor(s) on the cell surface.

Example 15—Peptide Compounds Significantly Reduced Brain Injury in a RatModel of Neonatal Hypoxic Ischemic Encephalopathy

Methods

P10 Wistar rat pups were subjected to unilateral right carotid ligation(Vannucci model) followed by 8% hypoxia. Experimental animals wereinjected with 150 mg/kg PA-dPEG24 (SEQ ID NO: 21) intraperitoneally.Controls included 1) cobra venom factor (CVF) injection to depletecomplement 2) therapeutic hypothermia for 6 hours. Animals wereharvested at 4, 8, 16 and 48 hours after intervention. CH50 assay wasperformed on rat plasma to measure systemic complement activity.Cerebral infarct volumes were measured by staining harvested braintissue with 2% TTC using image J software.

Results

Systemic complement activity was almost completely eliminated in animalsinjected with CVF for 72 hours after injection as expected. In thePA-dPEG24 (SEQ ID NO: 21) group, systemic complement activation wasdecreased by 70% at 0.5 hours after injection before gradually returningto baseline at 8 hours (FIG. 30). Hypoxia after carotid ligation yielded20% infarction. CVF injection before ligation/hypoxia almost completelyattenuated brain injury. HT reduces infarction by 50%. Injection withPA-dPEG24 (SEQ ID NO: 21) showed variable neuroprotection (FIG. 31) withan average reduction in infarction by 40%. Although not bound by anytheory, SEQ ID NO: 21 may mimic the neuroprotective effect oftherapeutic hypothermia by decreasing C1q deposition in the brain up to48 hours. There was significantly less C1q deposition in the SEQ ID NO:21 treated group (Normothermia+PIC1 (SEQ ID NO: 21)-triangle) whencompared to untreated HIE controls (Normothermia-diamond) at 4, 12, 24hours after brain injury (FIG. 32A).

SEQ ID NO: 21 also showed histological evidence of neuroprotection afterHIE. Brain histology five days after HIE showed the neuro-preservatoryaction of SEQ ID NO: 21 similar to therapeutic hypothermia. Neuronaldestruction was reduced in those treated with SEQ ID NO: 21, and thetreatment also increased viable cells in the brain (FIG. 32B). Forinstance, in FIG. 32B, Panels A-D show cresyl violet staining. Cresylviolet stains Nissl substance in neurons. The HIE group (Panel B) showsa significant decrease in cresyl violet staining compared to the SEQ IDNO: 21 group (Panel D), indicating the neuro-preservatory action of SEQID NO: 21 similar to therapeutic hypothermia (Panel C). Panels E-H showhematoxylin and eosin staining. Hematoxylin and Eosin stainingdemonstrated a greater degree of neuronal destruction (necrosis,pyknosis and karyorrhexis) in the HIE brain (Panels E, F, and G) whencompared to those treated with SEQ ID NO: 21 (Panel H). Panels I-L showacridine orange staining. Acridine orange stains viable parts of thebrain a bright green. HIE significantly decreases acridine orangestaining in the brain (Panel J). SEQ ID NO: 21 treatment restoresacridine orange staining, indicating the presence of more viable cellsin the brain (Panel L).

Histological neuroprotection also translated into functional improvementafter HIE. In a negative geotaxis test 1-7 days after HIE, animalsinjected with PIC1 (SEQ ID NO: 21) performed significantly better (FIG.32C, green bars) than normothermia HIE animals (FIG. 32C, red bars). Thenegative geotaxis test is an innate escape response test measuring thetime it takes for a pup to climb up when placed head down on a wiremesh.

This data showed that SEQ ID NO: 21 can reduce brain infarct volumeswithout prolonged systemic complement depletion, and improved functionaloutcomes after HIE. Although not bound by any theory, SEQ ID NO: 21 mayhave decreased C1q deposition in the brain. In some embodiments, SEQ IDNO: 21 is a useful adjunct therapy to HT to improve neurologicaloutcomes in HIE.

Example 16—PIC1 Inhibited Replication of Herpes Simplex Virus Type 1 andHerpes Simplex Virus Type 2

CV-1 cells were infected with HSV-1-GFP for 16 hours. Post-infection,CV-1 cells were detected by flow cytometry (FIG. 33). Uninfected cells(negative control, GFP−) result in a left peak, while the peak ininfected cells (GFP+) will shift to the right. When PA-PEG24 (SEQ ID NO:21) was added to the CV-1 cells before infection, it inhibited viralinfection in a dose dependent manner. Acyclovir (the standard of carefor severe HSV-1 infections) was also added as a control at the samemolar ratio. While Acyclovir was able to inhibit HSV-1 replicationsignificantly, it was never able to completely inhibit replication asindicated by the small GFP+ population of cells. This was in contrast toPA-PEG24 (SEQ ID NO: 21), where there was no detection of GFP+ cells at4-5 mM PA-PEG24 (SEQ ID NO: 21). In addition, PA-PEG24 (SEQ ID NO: 21)and Acyclovir showed minimal toxicity in CV-1 cells, which indicatedthat inhibition of viral replication was not due to cell death (FIG.33). PA-PEG24 (SEQ ID NO: 21) also inhibited replication of HSV-2 in adose dependent manner (FIG. 34).

Example 17—Peptide Compounds Promoted Growth of L. acidophilus and L.Leichmannii

The ability of PIC1 to not kill beneficial commensal Lactobacillus (asshown in FIG. 35) greatly enhances the safety of this antibiotic. Themost common side effects of current antibiotics areantibiotic-associated diarrhea and yeast infections because theantibiotics kill normal intestinal flora. Among the most dreadedantibiotic side effects is C. difficile colitis, which can belife-threatening and is often recurrent. FIG. 35 shows that PA-PEG24(SEQ ID NO: 21) can promote growth of Lactobacillus in a dose dependentmanner.

Example 18—PIC1 had Anti-Microbial Activity

Several PIC1 peptides were anti-bacterial across a broad range ofbacterial pathogens. For instance, PA-dPEG24 (also known as AF1, SEQ IDNO: 21) has been shown to inhibit growth of Staphylococcus aureus,Pseudomonas aeruginosa, and Klebsiella pneumoniae in a dose dependentmanner (as shown in FIG. 36A). Additionally, Pseudomonas aeruginosagrowth (FIG. 36B) was suppressed by five variants of PIC1 (AF1-5)peptides (SEQ ID NOs: 7, 8, 12, and 21. PIC1's (SEQ ID NO: 21)anti-bacterial activity was further confirmed by confocal microscopy,which showed PIC1 peptides bound to the outer surface of Staph aureusbacteria (FIG. 36C).

In some embodiments, the peptide compounds inhibited Neisseriagonorrhoeae growth in liquid culture. PA-PEG24 (SEQ ID NO: 21) reducedNeisseria gonorrhoeae growth at 20 mg (FIG. 37A) or 30 mg (FIG. 37B) ofPA-PEG24 (SEQ ID NO: 21) and demonstrated dose-dependent inhibition from0-30 mg/ml PA-PEG24 (SEQ ID NO: 21) (FIG. 37C). Additionally, colonycounts from Neisseria gonorrhoeae incubated with or without PA-PEG24(SEQ ID NO: 21) showed substantial reduction of Neisseria gonorrhoeaecolonies treated with 30 mg/ml of PA-PEG24 (SEQ ID NO: 21).

In conclusion, the data show that SEQ ID NO: 21, 5, 6, 7 and 8,respectively, had anti-bacterial activity against a broad range ofbacterial pathogens.

Example 19—PIC1 Inhibited Myeloperoxidase (MPO) Activity of Neutrophils

Myeloperoxidase (MPO) is an enzyme from neutrophils that createshypochlorite (bleach) in acute inflammation that damages invadingmicrobes and host cells alike. This enzyme is known to be destructive tohost tissues in Cystic Fibrosis (CF) and Hypoxic Ischemic Encephalopathy(HIE). PIC1(SEQ ID NO: 21) was found to inhibit MPO activity in sputumsamples (sol) isolated from cystic fibrosis (CF) patients. CF solsamples were incubated in the presence or absence of 20 mg/ml PIC1 (SEQID NO: 21) for 30 minutes followed by addition of TMB for 30 minutes atroom temperature (as shown in FIG. 38). MPO activity was then measuredby detection of TMB color change in a spectrophotometer at 450 nm. MPOactivity was found to be reduced in CF sol samples treated with PIC1.Preexisting MPO in the sputum of CF patients could have its enzymaticactivity blocked with the peptide compounds (FIG. 39 in a dose-dependentmanner (FIG. 39). Additionally, PA-PEG24 (SEQ ID NO: 21) was able toblock MPO enzymatic activity in a lysate of brain or brain membranepreparations of rats subject to hypoxic ischemic encephalopathy (asshown in FIGS. 40-42). MPO activity present in the lysates of purifiedhuman neutrophils can also be directly inhibited by PA-PEG24 (SEQ ID NO:21) (as shown in FIG. 42). FIG. 42 shows that PA-PEG24 (SEQ ID NO: 21)dose-dependently inhibits MPO activity in lysates of purified humanneutrophils (PMN). PMN lysates were incubated in the presence ofincreasing amounts of PA-PEG24 (SEQ ID NO: 21). MPO activity was thenmeasured by detection of TMB color change in a spectrophotometer at 450nm. Finally titration of the inhibition of purified MPO activity byPA-PEG24 (SEQ ID NO: 21) demonstrated that PA-PEG24 (SEQ ID NO: 21) candirectly inhibit MPO (as shown in FIG. 43). These findings demonstratedthat PIC1 has a surprising anti-inflammatory effect that is relevant toboth CF and HIE. This shows an alternative mechanism that could explainhow SEQ ID NO: 3-47 have anti-inflammatory activity.

Example 20—Acute Kidney Injury and Rhabdomyolysis: Peptide CompoundsInhibited Hemoglobin Production of Free Radical Activity

Peptide compounds inhibited the pseudo-peroxidase activity of hemoglobin(Hg) from human red blood cells. Hg, like myeloperoxidase (MPO),contains a heme group that can catalyze peroxide reactions affectingproteins, nucleic acids and lipids that damage host tissues. Thepseudo-peroxide activity of Hg is measured using a reaction betweenhydrogen peroxide (H₂O₂) and the chromogen tetramethylbenzidine (TMB).The oxidized TMB displays a color change that can be read in aspectrophotometer. Given that PA-PEG24 (SEQ ID NO: 21) could inhibit MPOperoxidase activity, we tested whether the peroxidase activity of Hgfrom lysed human RBCs could be inhibited by PA-PEG24 (SEQ ID NO: 21) inthe same type of experimental assay. To this end, RBC lysates yieldingfree Hg were incubated with TMB in absence or presence of PA-PEG24 (SEQID NO: 21). As shown in FIG. 44, increasing amounts of RBC lysates gavean increase in absorbance demonstrating increased oxidized TMBsubstrate. In the presence of 20 mg/ml PA-PEG24 (SEQ ID NO: 21), therewas no detectable oxidized TMB. In FIG. 45, increasing amounts ofPA-PEG24 (SEQ ID NO: 21) led to a dose-dependent decrease in oxidizedTMB signal. FIG. 46 shows a titration of oxidized TMB with increasingamounts of RBC lysate. In the presence of increasing amounts of PA-PEG24(SEQ ID NO: 21), oxidized TMB signal decreases dose dependently.Hemolysis leading to the presence of free Hg in human circulation istoxic to the kidneys, resulting in acute kidney toxicity and renalfailure. Thus, the data supported that PA-PEG24 (SEQ ID NO: 21) canprevent acute kidney injury by reducing hemolysis and pseudo-peroxidaseactivity caused by the release of free hemoglobin. Thus, the peptidescan prevent acute kidney injury, as well as treat or prevent hemolyticconditions such as, e.g., sickle cell disease, hemolytic transfusionreaction, autoimmune hemolysis, etc.

To compare the activity of IVIG versus PIC1, animals were treatedprophylactically with an equal dose of IVIG or PIC1 (40 mg/animal) orsaline control. Blood of animals that received IVIG or PIC1 beforexenotransfusion were subject to two-color flow cytometry probing forboth F human glycophorin A expression and bound C3 fragments.Representative flow cytometry plots of RBCs at 0.5, 5, and 20 minutespost-transfusion are shown for IVIG (FIGS. 27A-C) and PIC1 (FIG. 27D-F)animals. FIG. 28A shows hemolysis measured as free Hb for animalsreceiving IVIG or PIC1 prophylactically compared to saline control. At30 seconds after transfusion, PIC1-treated animals demonstratedincreased numbers of C3-negative human RBCs (p 5 0.05) and a 3.6-fold (p5 0.04) decrease in C3-opsonized human RBCs compared with IVIG treatedanimals (FIG. 28B). At 30 seconds after transfusion, PIC1-treatedanimals showed a trend toward decreased C3 opsonization of nonhuman(glycophorin A-negative) RBCs (p 5 0.08) compared with IVIG animals(FIG. 28C). Total surviving human RBCs at 20 minutes was increased1.6-fold (p 5 0.03) for PIC1-treated compared with IVIG treated animals(FIG. 28D).

To ascertain if the intravascular toxic free Hb would cause acute kidneyinjury, the animals were monitored for 6 hours post-transfusion. In theIVIG prophylaxis group, two of the three animals were noted to exhibitgross hematuria as well as three of four animals in the saline group,but the urine of the PIC1-treated animals appeared normal. At necropsy,the kidneys of the IVIG group looked darker and enlarged compared to thekidneys of the PIC1-treated group, which appeared normal (FIG. 29B). Onmeasuring the weight of the kidneys individually, the saline groupexhibited a significant increase in weight compared to animals receivingPIC1 (p 5 0.0001) whereas the IVIG group also demonstrated a significantincrease in weight compared to animals receiving PIC1 (p 5 0.005),consistent with edematous kidneys (FIG. 29A). Histopathology sectionsrevealed that the saline and IVIG-treated animals had early acutetubular necrosis, as noted by the disappearing nucleus and nuclearmaterial and tubular edema (saline, FIG. 29C, i-ii; IVIG prophylaxis,FIG. 29C, iii-iv) compared to the slight edema, but otherwise healthykidney architecture for the PIC1 animals (PIC1 prophylaxis, FIG. 29C, vand vi). Taken together, these data demonstrated that PIC1 inhibitshemolysis of the transfused mismatched RBCs preventing subsequent freeHb-associated acute kidney injury.

Rhabdomyolysis is a serious syndrome caused by direct or indirect muscleinjury resulting from crush injuries, certain infections, use of certainmedications, seizures and other events. The resulting injury to tissuescauses release of myoglobin, the oxygen-binding protein found in muscletissue, into the bloodstream. Myoglobin is harmful to kidneys and thereis currently no treatment for Rhabdomyolysis. The discovery thatPA-PEG24 (SEQ ID NO: 21) can inhibit pseudo-peroxidase activity ofhemoglobin (Hg) showed that PA-PEG24 (SEQ ID NO: 21) is efficacious intreating rhabdomyolysis. Because hemoglobin is similar to myoglobin,PA-PEG24's (SEQ ID NO: 21) ability to inhibit hemoglobin suggests itsefficacy in inhibiting the peroxidase activity of myoglobin.

Example 21—Toxicity Data for PIC1 Peptides and Variants

Single-dose toxicology studies with PA-PEG24 (SEQ ID NO: 21) in rats forboth maximum deliverable dose and upper limit of the effective doserange have been performed. For the maximum deliverable dose (limited bymaximum achievable concentration of PA-PEG24 (SEQ ID NO: 21) (60 mg [240mg/kg] i.v.)) and deliverable volume, in the treatment group, multipleblood draws were performed on six animals for 14 days. No toxicitieswere seen on the blood work (CBC and complete blood chemistry) over thefirst 24 hours, 48 hours, day 7 or day 14. A small drop in hemoglobin isnoted at 48 hours due to multiple blood draws taken during the first 24hours. Normal histology was found for in the brain, heart, lungs, liver,spleen, kidneys, and pancreas. The most pertinent blood parameters forthis experiment are shown in TABLE 11.

TABLE 11 Max deliverable PIC1: toxicology. Pretreatment PIC1 48 Hr PIC1Day 7 PIC1 Day 14 (n = 6) (n = 6) (n = 6) (n = 6) Units WBC 5.5 (±1.6)10.5 (±2.9) 5.4 (±1.12) N/A 10³/mL RBC 6.2 (±0.3) 5.3 (±0.3) 5.8 (±0.5)N/A 10⁶/μL Platelets 1004 (±130) 1045 (±156) 623 (±417) N/A 10³/μL AST98.2 (±15.4) 69.0 (±8.7) 84.3 (±6.74) 104 (±15) U/L ALT 76.3 (±20.0)44.0 (±10.4) 76.3 (±10.9) 55.8 (±5.6) U/L AlkPhos 150 (±29) 194 (±29)147 (±34) 154 (±42) U/L Bilirubin 0.1 (±0) 0.1 (±0) 0.1 (±0) 0.1 (±0)mg/dL GGT 1.2 (±0.4) 1 (±0) 1 (±0) 1 (±0) U/L BUN 12.3 (±2.3) 15.8(±2.1) 13.5 (±1.64) 14.7 (±1.0) mg/dL Creatinine 0.3 (±0.1) 0.3 (±0) 0.4(±0.1) 0.4 (±0.1) mg/dL Lipase 10.3 (±2.0) 10.3 (±1.5) 8.5 (±1.05) 9.7(±2.0) U/L CPK 454 (±200) 142 (±47) 316 (±90) 580 (±172) U/L

Toxicity studies were later performed with larger numbers of animals(n=18 in the treatment group) using a high-dose at the upper end of thetherapeutic range (PA-PEG24 (SEQ ID NO: 21) at 40 mg [160 mg/kg] i.v.).Eight animals were sacrificed at day 3, and ten were sacrificed at day14. Blood chemistry assay results (CBC, superchem, and coagulation)showed no differences between pretreatment, day 3 or day 14 values(TABLE 12). There was no histological evidence for toxicity in any organ(including brain, heart, lungs, liver, spleen, kidneys, and pancreas) ateither time point. Thus, single-dose toxicological evaluation of SEQ IDNO: 21 revealed no safety concerns.

TABLE 12 High-dose PIC1: toxicology. Pretreatment PIC1 48 Hr PIC1 Day 14(n = 18) (n = 8) (n = 10) Units WBC 6.6 (±1.4) 6.0 (±3.0) 7.3 (±2.5)10³/mL RBC 6.5 (±1.4) 5.8 (±0.5) 6.6 (±0.4) 10⁶/μL Platelets 927 (±195)987 (±93) 825 (±229) 10³/μL AST 102 (±16) 108 (±26) 111 (±14) U/L ALT62.1 (±12.1) 62.8 (±4.0) 77.4 (±13.9) U/L AlkPhos 142 (±59) 104 (±19)115 (±56) U/L Bilirubin 0.1 (±0) 0.1 (±0) 0.1 (±0) mg/dL GGT 1.0 (±0)1.0 (±0) 1.1 (±0.4) U/L BUN 10.9 (±2.0) 17.8 (±3.2) 13.2 (±1.9) mg/dLCreatinine 0.3 (±0) 0.3 (±0) 0.3 (±0) mg/dL Lipase 5.9 (±1.1) 11.8(±1.5) 6.4 (±1.3) U/L CPK 544 (±208) 538 (±128) 592 (±163) U/L

What is claimed is:
 1. A peptide comprising the amino acid sequence ofany one of SEQ ID NOS: 5, 6, 7, 8, 11, 12, and
 31. 2. A pharmaceuticalcomposition comprising a therapeutically effective amount of the peptideof claim 1 and at least one pharmaceutically acceptable carrier,diluent, or excipient.
 3. The peptide of claim 1 comprising the aminoacid sequence set forth in SEQ ID NO:
 11. 4. A pharmaceuticalcomposition comprising a therapeutically effective amount of the peptideof claim 3 and at least one pharmaceutically acceptable carrier,diluent, or excipient.
 5. The peptide of claim 1 comprising the aminoacid sequence of SEQ ID NO:
 5. 6. The peptide of claim 1 comprising theamino acid sequence of SEQ ID NO:
 6. 7. The peptide of claim 1comprising the amino acid sequence of SEQ ID NO:
 7. 8. The peptide ofclaim 1 comprising the amino acid sequence of SEQ ID NO:
 8. 9. Thepeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 11.10. The peptide of claim 1 comprising the amino acid sequence of SEQ IDNO:
 12. 11. The peptide of claim 1 comprising the amino acid sequence ofSEQ ID NO: 31.